Molecular profiling for cancer

ABSTRACT

Provided herein are methods and systems of molecular profiling of diseases, such as cancer. In some embodiments, the molecular profiling can be used to identify treatments for a disease, such as treatments that were not initially identified as a treatment for the disease or not expected to be a treatment for a particular disease. The cancer can be an ovarian cancer.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication 61/427,788, filed on Dec. 28, 2010; which application isincorporated herein by reference in its entirety.

This application claims the benefit of U.S. patent application Ser. No.12/658,770, filed Feb. 12, 2010; which application claims the benefit ofprovisional patent application 61/151,758, filed on Feb. 11, 2009; U.S.provisional patent application 61/170,565, filed on Apr. 17, 2009; U.S.provisional patent application 61/217,289, filed May 28, 2009; U.S.provisional patent application 61/229,686, filed on Jul. 29, 2009; U.S.provisional patent application 61/279,970, filed Oct. 27, 2009; U.S.provisional patent application 61/261,709, filed Nov. 16, 2009; and U.S.provisional patent application 61/294,440, filed Jan. 12, 2010; andfurther claims the benefit of U.S. patent application Ser. No.12/579,241, filed on Oct. 14, 2009, which claims the benefit of U.S.provisional application 61/105,335, filed on Oct. 14, 2008, and U.S.provisional patent application 61/106,921, filed on Oct. 20, 2008; andfurther claims the benefit of U.S. patent application Ser. No.11/750,721, filed on May 18, 2007, which claims the benefit of U.S.provisional application 60/747,645, filed on May 18, 2006; all of whichapplications are incorporated herein by reference in their entirety.

BACKGROUND

Disease states in patients are typically treated with treatment regimensor therapies that are selected based on clinical based criteria; thatis, a treatment therapy or regimen is selected for a patient based onthe determination that the patient has been diagnosed with a particulardisease (which diagnosis has been made from classical diagnosticassays). Although the molecular mechanisms behind various disease stateshave been the subject of studies for years, the specific application ofa diseased individual's molecular profile in determining treatmentregimens and therapies for that individual has been disease specific andnot widely pursued.

Some treatment regimens have been determined using molecular profilingin combination with clinical characterization of a patient such asobservations made by a physician (such as a code from the InternationalClassification of Diseases, for example, and the dates such codes weredetermined), laboratory test results, x-rays, biopsy results, statementsmade by the patient, and any other medical information typically reliedupon by a physician to make a diagnosis in a specific disease. However,using a combination of selection material based on molecular profilingand clinical characterizations (such as the diagnosis of a particulartype of cancer) to determine a treatment regimen or therapy presents arisk that an effective treatment regimen may be overlooked for aparticular individual since some treatment regimens may work well fordifferent disease states even though they are associated with treating aparticular type of disease state.

Patients with refractory or metastatic cancer are of particular concernfor treating physicians. The majority of patients with metastatic orrefractory cancer eventually run out of treatment options or may suffera cancer type with no real treatment options. For example, some patientshave very limited options after their tumor has progressed in spite offront line, second line and sometimes third line and beyond) therapies.For these patients, molecular profiling of their cancer may provide theonly viable option for prolonging life.

More particularly, additional targets or specific therapeutic agents canbe identified assessment of a comprehensive number of targets ormolecular findings examining molecular mechanisms, genes, gene expressedproteins, and/or combinations of such in a patient's tumor. Identifyingmultiple agents that can treat multiple targets or underlying mechanismswould provide cancer patients with a viable therapeutic alternative on apersonalized basis so as to avoid standard therapies, which may simplynot work or identify therapies that would not otherwise be considered bythe treating physician.

There remains a need for better theranostic assessment of cancervictims, including molecular profiling analysis that identifies one ormore individual profiles to provide more informed and effectivepersonalized treatment options, resulting in improved patient care andenhanced treatment outcomes. The present invention provides methods andsystems for identifying treatments for these individuals by molecularprofiling one or more sample from the individual.

SUMMARY OF THE INVENTION

The present invention provides methods and system for molecularprofiling, using the results from molecular profiling to identifytreatments for individuals. In some embodiments, the treatments were notidentified initially as a treatment for the disease.

In an aspect, the invention provides a method of identifying a candidatetreatment for a subject in need thereof, comprising: (a) determining amolecular profile for one or more sample from the subject on a panel ofgene or gene products, wherein the molecular profile comprises theresults of assessing the panel of gene or gene products by: i)performing immunohistochemistry (IHC) analysis on the one or more samplefrom the subject on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or moreof: AR, BCRP, CAV-1, CD20, CD52, CK 5/6, CK14, CK17, c-kit, CMET, COX-2,Cyclin D1, E-Cad, EGFR, ER, ERCC1, HER-2, IGF1R, Ki67, MGMT, MRP1, P53,p95, PDGFR, PGP, PR, PTEN, RRM1, SPARC, TLE3, TOPO1, TOPO2A, TS andTUBB3; ii) performing microarray analysis on the one or more sample on1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 or more of:ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA,CES2, cKit, c-MYC, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2,ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1,HER2/ERBB2, HIF1A, HSP90, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, LCK, LYN,MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC,PDGFRa, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1,RRM2, RRM2B, RXRB, RXRG, SIK2, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5,SPARC, TK1, TNF, TOP2B, TOP2A, TOPO1, TXNRD1, TYMS, VDR, VEGFA, VHL,YES1, and ZAP70; iii) performing fluorescent in-situ hybridization(FISH) analysis on the one or more sample on 1, 2, 3, 4, 5, 6 or 7 of:ALK, cMET, c-MYC, EGFR, HER-2, PIK3CA, and TOPO2A; and iv) performingDNA sequence analysis or PCR on the one or more sample on 1, 2, 3, 4, 5or 6 of: BRAF, c-kit, EGFR, KRAS, NRAS, and PIK3CA; (b) comparing themolecular profile of the subject to a molecular profile of a referenceto identify which of the members of the panel are differentiallyexpressed between the one or more sample and the reference; and (c)identifying a treatment that is associated with one or more members ofthe panel are differentially expressed between the one or more sampleand the reference, thereby identifying the candidate treatment.

In another aspect, the invention provides a method of method ofidentifying a candidate treatment for an ovarian cancer in a subject inneed thereof, comprising: (a) determining a molecular profile for one ormore sample from the subject on a panel of gene or gene products,wherein the molecular profile comprises the results of assessing thepanel of gene or gene products by: i) performing an immunohistochemistry(IHC) analysis on a sample from the one or more subject on 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more of: AR, ER, ERCC1, HER2, MGMT, PGP, PR, PTEN,RRM1, SPARC, TLE3, TOP2A, TOPO1, TS; ii) performing a microarrayanalysis on the one or more sample on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20 or more of: BRCA1, BRCA2, DHFR, ER, ERCC1, GART, HIF-1α, IGFBP3,IGFBP4, IGFBP5, MGMT, P-gp (ABCB1), PR, RRM1, RRM2, RRM2B, SPARC, SRC,TOPO I, TOPO IIα, TOPO IIβ, TS (TYMS), VDR, VEGFR1 (FLT1), VEGFR2 (KDR),VHL; iii) performing a fluorescent in-situ hybridization (FISH) analysison the one or more sample on HER2; (b) comparing the molecular profileof the subject to a molecular profile of a reference to identify whichof the members of the panel are differentially expressed between the oneor more sample and the reference; and (c) identifying a treatment thatis associated with one or more members of the panel are differentiallyexpressed between the one or more sample and the reference, therebyidentifying the candidate treatment. In some embodiments, the methodfurther comprises performing (IHC) analysis on a sample from the subjecton 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of: BCRP, CAV-1, CD20, CD52, CK5/6, CK14, CK17, c-kit, CMET, COX-2, Cyclin D1, E-Cad, EGFR, IGF1R,Ki67, MRP1, P53, p95, PDGFR and TUBB3. In addition, the method canfurther comprise performing microarray analysis on the sample on 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more of: ABCC1,ABCG2, ADA, AR, ASNS, BCL2, BIRC5, CD33, CD52, CDA, CES2, cKit, c-MYC,DCK, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERCC3, FOLR2, FYN,GNRH1, GSTP1, HCK, HDAC1, HER2/ERBB2, HSP90, LCK, LYN, MET, MIH1, MS4A1,MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRa, PDGFRA, PDGFRB,POLA1, PTEN, PTGS2, RAF1, RARA, RXRB, RXRG, SIK2, SSTR1, SSTR2, SSTR3,SSTR4, SSTR5, TK1, TNF, TXNRD1, VEGFA, YES1, and ZAP70. The fluorescentin-situ hybridization (FISH) analysis on the sample can also beperformed on 1, 2, 3, 4, 5 or 6, of: ALK, cMET, c-MYC, EGFR, PIK3CA, andTOPO2A. For example, the FISH analysis can be performed for EGFR. Insome embodiments, the method further comprises performing DNA sequenceanalysis or PCR on the sample on 1, 2, 3, 4, 5 or 6 of: BRAF, c-kit,EGFR, KRAS, NRAS, and PIK3CA. As appropriate, the method can furthercomprise all of these additional analyses.

The molecular techniques can be performed on a single sample or onmultiple samples from a subject, e.g., on one tumor sample and on oneblood sample. The molecular techniques can be performed in any order. Incases where the sample does not pass a quality test, one or moretechnique may not be performed.

In some embodiments of the methods of the invention, identifying atreatment that is associated with one or more members of the panel aredifferentially expressed comprises: (a) correlating the one or moremembers of the panel are differentially expressed with a set of rules,wherein the set of rules comprises a mapping of treatments whosebiological activity is determined against cancer cells that havedifferent level of, overexpress, underexpress, and/or have mutations inone or more members of the panel of gene or gene products; and (b)identifying the treatment based on the correlating in (a). The set ofrules can include one or more of the rules listed in Table 4 and/orTable 5. For example, the set of rules can comprise at least 5, 10, 25,50 or 100 rules in Table 5. In some embodiments, the set of rulescomprises all of the rules in Tables 4 or 5. The mapping of treatmentscontained within the set of rules can be based on the efficacy ofvarious treatments particular for a target gene or gene product thereof.The mapping of treatments that are associated with one or more membersof the panel can be listed in Table 11 or Table 12.

In some embodiments of the methods of the invention, the one or moresample comprises formalin-fixed paraffin-embedded (FFPE) tissue, freshfrozen (FF) tissue, or tissue comprised in a solution that preservesnucleic acid or protein molecules. The one or more sample can includewithout limitation a fixed tissue, an unstained slide, a bone marrowcore or clot, a core needle biopsy, a bodily fluid, a malignant fluid, afine needle aspirate (FNA), or a combination of any thereof. The samplecan comprise diseased tissue such as a tumor tissue. The sample caninclude diseased cells such as cancer cells. The sample may comprisecells from any tissue of the body, e.g., the cells can be selected fromthe group consisting of adipose, adrenal cortex, adrenal gland, adrenalgland—medulla, appendix, bladder, blood, blood vessel, bone, bonecartilage, brain, breast, cartilage, cervix, colon, colon sigmoid,dendritic cells, skeletal muscle, enodmetrium, esophagus, fallopiantube, fibroblast, gallbladder, kidney, larynx, liver, lung, lymph node,melanocytes, mesothelial lining, myoepithelial cells, osteoblasts,ovary, pancreas, parotid, prostate, rectum, salivary gland, sinustissue, skeletal muscle, skin, small intestine, smooth muscle, stomach,synovium, joint lining tissue, tendon, testis, thymus, thyroid, uterus,and uterus corpus. The bodily fluid can include peripheral blood, sera,plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen (including prostatic fluid),Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecalmatter, hair, tears, cyst fluid, pleural and peritoneal fluid,pericardial fluid, malignant effusion, lymph, chyme, chyle, bile,interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates or other lavage fluids. Insome embodiments, the one or more sample comprises one or more of amicrovesicle population, a microRNA and a circulating biomarker. Thebiomarkers assessed can be associated with the microvesicle population,e.g., as a surface marker or as internal payload of a vesicle.

In embodiments of the methods of the invention, the reference is from anon-cancerous sample. The reference can be from the subject, or thereference can be from another subject or group of subjects, e.g.,another subject or group of subjects that do not have the cancer. Whenthe reference is from the subject, the reference may comprise anon-diseased sample, e.g., normal adjacent tissue, or the reference maybe from a different time point, such as at an earlier time point. Thereference can derived from a plurality of reference samples. Forexample, the reference can be an average profile from a number ofnon-cancerous samples. In another embodiment, the reference comprisesprofiles from different individuals for different biomarkers.

In embodiments of the methods of the invention, the IHC analysis isperformed on at least 5, or 15 of the biomarkers listed above. The IHCanalysis can be performed on all of the biomarkers listed above. Inembodiments of the methods of the invention, the microarray analysis isperformed on at least 5, 10, 15, 20, or 30 of the biomarkers listed. Themicroarray analysis can be performed on all of the biomarkers listedabove. Similarly, the sequencing, PCR and/or FISH can be performed onall of the biomarkers listed above. In embodiments of the methods of theinvention, the all members of the panel of genes or gene products listedabove are assessed.

In embodiments of the methods of the invention, the microarray analysiscan be a low density microarray, an expression microarray, a comparativegenomic hybridization (CGH) microarray, a single nucleotide polymorphism(SNP) microarray, a proteomic array or an antibody array. Any usefulcombination of array techniques can be used. The low density microarraycan be a PCR-based microarray, such as a Taqman™ Low Density Microarray(Applied Biosystems, Foster City, Calif.).

The panel of gene or gene products assessed according to the subjectmethods can include without limitation one or more of ABCC1, ABCG2,ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1, beta IIItubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33,CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK 17, CK5/6, c-KIT, c-Met, c-Myc, COX-2, Cyclin D1, DCK, DHFR, DNMT1, DNMT3A,DNMT3B, E-Cadherin, ECGF1, EGFR, EML4-ALK fusion, EPHA2, Epiregulin, ER,ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2,FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1,hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R,IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT,K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN, MET, MGMT, MLH1, MMR,MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, NRAS, ODC1, OGFR,p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR,PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, PTPN12, RAF1, RARA,RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3,SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS, TUBB3,TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, and ZAP70. In anembodiment, the panel of gene or gene products comprises one or moregene or gene product in Table 2. Any of the genes and gene productsthereof can be assessed using one or more molecular technique asdescribed herein or known in the art. The genes and gene productsthereof can include any gene or gene product whose status can beassociated with benefit of a candidate treatment, a lack of benefit of acandidate treatment, or a prognosis. The invention is not only limitedto the candidate treatments that are currently known, but alsocontemplates analysis of other genes or gene products thereof that arelinked to existing or novel treatments in the future as well.

In embodiments of the methods of the invention, the microarray analysiscomprises identifying whether a gene is upregulated or downregulatedrelative to a reference with statistical significance. The statisticalsignificance can be determined at a set p-value, e.g., a p-value of lessthan or equal to 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. In someembodiments, the p-value is corrected for multiple comparisons, e.g.,using a false discovery rate, Bonneferoni's correction or a modificationthereof.

The IHC analysis performed per the methods of the invention can comprisedetermining whether 30% or more of at least a portion of the one or moresample is +2 or greater in staining intensity. The sample can comprise atumor such that the IHC comprises determining whether 30% or more of atleast a portion of a tumor sample is +2 or greater in stainingintensity.

In embodiments of the methods of the invention, a list of multiplecandidate treatments is identified. One or more candidate treatments canbe identified for more than one of the genes or gene products that areassessed. The list of candidate treatments can be prioritized. In someembodiments, the prioritizing comprises ordering the treatments fromhigher priority to lower priority according to treatments based onmicroarray analysis and either IHC or FISH analysis; treatments based onIHC analysis but not microarray analysis; and treatments based onmicroarray analysis but not IHC analysis. In some embodiments,on-compendium treatments are prioritized over non-compendium treatments.The priority can depend on a prognosis. The prognosis can guideselection of the candidate treatment, e.g., a more aggressive therapycan be selected for a cancer with a worse prognosis, or a lessaggressive treatment can be selected for cancer with a better prognosis.

The candidate treatment identified by the methods of the invention caninclude one or more therapeutic agent. The therapeutic agent can be acytotoxic agent, a cytostatic agent, an immunomodulatory agent, a drug,a pharmaceutical agent, a small molecule, a protein therapy, an antibodyor fragment thereof, a viral therapy agent, a gene therapy agent, achemotherapeutic agent, a hormonal therapy, a radiotherapy, animmunotherapy, or any combination thereof. The one or more therapeuticagent can be selected from those listed in Table 5, Table 11 or Table12.

In embodiments of the methods of the invention, the subject has a newlydiagnosed disease. In other embodiments, the subject has been previouslytreated with the candidate treatment. Alternately, the methods areperformed wherein the subject has not previously been treated with thecandidate treatment. The subject may have been previously treated forthe cancer. The cancer can be a metastatic cancer. The cancer can be arecurrent cancer. The cancer can be refractory to one or more priortreatment. In some embodiments, the prior treatment comprises thestandard of care for the cancer.

The cancer that is profiled according to the subject methods can be anovarian cancer. In some embodiments, the ovarian cancer comprises anovarian surface epithelium carcinoma (EOC). The EOC can be withoutlimitation a surface epithelial tumor, serous cancer, mucinous cancer,endometriod cancer, clear cell cancer, carcinosarcoma, Brenner tumor,cancer of the fallopian tubes, or a female peritoneal cancer. Theovarian cancer can be a non-epithelium ovarian carcinoma (non-EOC). Thenon-EOC can be without limitation a sarcoma of the ovary, malignant germcell tumor, sex cord-stromal tumor, gonadoblastoma, lymphoma, or otherrare tumor of the ovary.

The methods of the invention can also be used to profile a cancerselected from the group consisting of an acute lymphoblastic leukemia;acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer;AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas;atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer;brain stem glioma; brain tumor, brain stem glioma, central nervoussystem atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma; breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site (CUP);carcinoid tumor; carcinoma of unknown primary site; central nervoussystem atypical teratoid/rhabdoid tumor; central nervous systemembryonal tumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer;medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma;Merkel cell skin carcinoma; mesothelioma; metastatic squamous neckcancer with occult primary; mouth cancer; multiple endocrine neoplasiasyndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm;mycosis fungoides; myelodysplastic syndromes; myeloproliferativeneoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma;Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lungcancer; oral cancer; oral cavity cancer; oropharyngeal cancer;osteosarcoma; other brain and spinal cord tumors; ovarian cancer;ovarian epithelial cancer; ovarian germ cell tumor; ovarian lowmalignant potential tumor; pancreatic cancer; papillomatosis; paranasalsinus cancer; parathyroid cancer; pelvic cancer; penile cancer;pharyngeal cancer; pineal parenchymal tumors of intermediatedifferentiation; pineoblastoma; pituitary tumor; plasma cellneoplasm/multiple myeloma; pleuropulmonary blastoma; primary centralnervous system (CNS) lymphoma; primary hepatocellular liver cancer;prostate cancer; rectal cancer; renal cancer; renal cell (kidney)cancer; renal cell cancer; respiratory tract cancer; retinoblastoma;rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small celllung cancer; small intestine cancer; soft tissue sarcoma; squamous cellcarcinoma; squamous neck cancer; stomach (gastric) cancer;supratentorial primitive neuroectodermal tumors; T-cell lymphoma;testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroidcancer; transitional cell cancer; transitional cell cancer of the renalpelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer;uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer;Waldenstrom macroglobulinemia; a Wilm's tumor; or any combinationthereof. In some embodiments, the cancer comprises a cancer of unknownprimary (CUP).

The methods of the invention can be used to determine a prognosis forthe cancer based on the molecular profiling comparison. The prognosiscan guide selection of the candidate treatment, e.g., a more aggressivetherapy can be selected for a cancer with a worse prognosis, or a lessaggressive treatment can be selected for cancer with a better prognosis.The prognosis may be based on analysis of one or more of cMet, IGF1R,Class III beta tubulin (TUBB3), PIK3CA, and/or the biomarkers in Table16 herein. Any molecular techniques herein or known in the art can beused to assess prognostic markers. In some embodiments, cMET is assessedby IHC and/or FISH. In other embodiments, IGF1R is assessed by IHC.Class III beta tubulin can be assessed by IHC. PIK3CA can be assessed byFISH.

The methods of invention can provide patient benefit. In someembodiments, progression free survival (PFS) or disease free survival(DFS) for the subject is extended by selection of the candidatetreatment. The subject's lifespan can be extended by the candidatetreatment.

In another aspect, the invention provides a system for carrying out themethod of any previous claim, comprising: a host server; a userinterface for accessing the host server to access and input data; aprocessor for processing the inputted data; a memory coupled to theprocessor for storing the processed data and instructions for: i)accessing the molecular profile generated for the one or more sample;ii) determining which of the members of the panel are differentiallyexpressed between the one or more sample and the reference; and iii)accessing a rules database to identify one or more agent that interactswith the members of the panel that were determined to be differentiallyexpressed between the one or more sample and the reference; and adisplay means for displaying the members of the panel that weredetermined to be differentially expressed between the one or more sampleand the reference and the agents that are associated with them. In thesystems of the invention, the rules database can comprise one or more ofthe rules in Tables 4 or 5. For example, the system can comprise atleast 5, 10, 25, 50 or 100 rules in Table 5. In some embodiments, therules database comprises all of the rules in Tables 4 or 5.

In another aspect, the invention provides a method of generating a setof evidence-based associations, comprising: (a) searching one or moreliterature database by a computer using an evidence-based medicinesearch filter to identify articles comprising a gene or gene productthereof, a disease, and one or more therapeutic agent; (b) filtering thearticles identified in (a) to compile evidence-based associationscomprising the expected benefit and/or the expected lack of benefit ofthe one or more therapeutic agent for treating the disease given thestatus of the gene or gene product; (c) adding the evidence-basedassociations compiled in (b) to the set of evidence-based associations;and (d) repeating steps (a)-(c) for an additional gene or gene productthereof. The status of the gene can include one or more assessments asdescribed herein which relate to a biological state, e.g., one or moreof an expression level, a copy number, and a mutation. The genes or geneproducts thereof can be one or more genes or gene products thereofselected from Table 2. For example, the method can be repeated for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more of the genes orgene products thereof in Table 2. The genes or gene products thereof canalso comprise all genes or gene products thereof in any one of Table 2,Table 10, Table 11, and Table 12. The disease can be a disease describedhere, e.g., in embodiment the disease comprises an ovarian cancer. Theone or more literature database can be selected from the groupconsisting of the National Library of Medicine's (NLM's) MEDLINE™database of citations, a patent literature database, and a combinationthereof. The evidence-based medicine filter can be selected from thegroup consisting of a generic evidence-based medicine filter, a McMasterUniversity optimal search strategy evidence-based medicine filter, aUniversity of York statistically developed search evidence-basedmedicine filter, and a University of California San Francisco systemicreview evidence-based medicine filter. The filtering in (b) can beperformed at least in part by one or more expert. The one or more expertcan be a trained scientist or physician. In embodiments, the set ofevidence-based associations comprise one or more of the rules in Table5. For example, the set of evidence-based associations can include atleast 5, 10, 25, 50 or 100 rules in Table 5. In some embodiments, theset of evidence-based associations comprises or consists of all of therules in Table 5.

In an aspect, the invention provides a computer readable mediumcomprising the set of evidence-based associations generated by thesubject methods. The invention further provides a computer readablemedium comprising one or more rules in Table 5. In an embodiment, thecomputer readable medium comprises at least 5, 10, 25, 50 or 100 rulesin Table 5. For example, the computer readable medium can comprise allrules in Table 5.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are used, and the accompanying drawings ofwhich:

FIG. 1 illustrates a block diagram of an illustrative embodiment of asystem for determining individualized medical intervention for aparticular disease state that uses molecular profiling of a patient'sbiological specimen that is non disease specific.

FIG. 2 is a flowchart of an illustrative embodiment of a method fordetermining individualized medical intervention for a particular diseasestate that uses molecular profiling of a patient's biological specimenthat is non disease specific.

FIGS. 3A through 3D illustrate an illustrative patient profile report inaccordance with step 80 of FIG. 2.

FIG. 4 is a flowchart of an illustrative embodiment of a method foridentifying a therapeutic agent capable of interacting with a target.

FIGS. 5-14 are flowcharts and diagrams illustrating various parts of aninformation-based personalized medicine drug discovery system and methodin accordance with the present invention.

FIGS. 15-25 are computer screen print outs associated with variouscomponents of the information-based personalized shown in FIGS. 5-14.

FIGS. 26A-26H represent a table that shows the frequency of asignificant change in expression of gene expressed proteins by tumortype.

FIGS. 27A-27H represent a table that shows the frequency of asignificant change in expression of certain genes by tumor type.

FIGS. 28A-28O represent a table that shows the frequency of asignificant change in expression for certain gene expressed proteins bytumor type.

FIG. 29 is a table which shows biomarkers (gene expressed proteins)tagged as targets in order of frequency based on FIG. 28.

FIGS. 30A-30O represent a table that shows the frequency of asignificant change in expression for certain genes by tumor type.

FIG. 31 is a table which shows genes tagged as targets in order offrequency based on FIG. 30.

FIG. 32 illustrates progression free survival (PFS) using therapyselected by molecular profiling (period B) with PFS for the most recenttherapy on which the patient has just progressed (period A). IfPFS(B)/PFS(A) ratio ≧1.3, then molecular profiling selected therapy wasdefined as having benefit for patient.

FIG. 33 is a schematic of methods for identifying treatments bymolecular profiling if a target is identified.

FIG. 34 illustrates the distribution of the patients in the study asperformed in Example 1.

FIG. 35 is graph depicting the results of the study with patients havingPFS ratio ≧1.3 was 18/66 (27%).

FIG. 36 is a waterfall plot of all the patients for maximum % change ofsummed diameters of target lesions with respect to baseline diameter.

FIG. 37 illustrates the relationship between what clinician selected aswhat she/he would use to treat the patient before knowing what themolecular profiling results suggested. There were no matches for the 18patients with PFS ratio ≧1.3.

FIG. 38 is a schematic of the overall survival for the 18 patients withPFS ratio ≧1.3 versus all 66 patients.

FIG. 39 illustrates a molecular profiling system that performs analysisof a cancer sample using a variety of components that measure expressionlevels, chromosomal aberrations and mutations. The molecular “blueprint”of the cancer is used to generate a prioritized ranking of druggabletargets and/or drug associated targets in tumor and their associatedtherapies.

FIG. 40 shows an example output of microarray profiling results andcalls made using a cutoff value.

FIGS. 41A-41L illustrate an illustrative patient report based onmolecular profiling of an ovarian cancer.

FIGS. 42A-42L illustrate another illustrative patient report based onmolecular profiling of an ovarian adenocarcinoma.

FIGS. 43A-B illustrate a workflow chart for identifying a therapeuticfor an individual having breast cancer. The workflow of FIG. 43A feedsinto the workflow of FIG. 43B as indicated.

FIGS. 44A-B illustrates biomarkers used for identifying a therapeuticfor an individual having breast cancer such as when following theworkflow of FIG. 43. FIG. 44A illustrate a biomarker centric view of theworkflow described above in different cancer settings. FIG. 44Billustrates additional biomarkers assessed depending on the criteriashown.

FIG. 45 illustrates the percentage of HER2 positive breast cancers thatare likely to respond to treatment with trastuzumab (Herceptin®), whichis about 30%. Characteristics of the tumor that can be identified bymolecular profiling are shown as well.

FIG. 46 illustrates a diagram showing a biomarker centric (FIG. 46A) andtherapeutic centric (FIG. 46B) approach to identifying a therapeuticagent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and systems for identifyingtherapeutic agents for use in treatments on an individualized basis byusing molecular profiling. The molecular profiling approach provides amethod for selecting a candidate treatment for an individual that couldfavorably change the clinical course for the individual with a conditionor disease, such as cancer. The molecular profiling approach providesclinical benefit for individuals, such as identifying drug target(s)that provide a longer progression free survival (PFS), longer diseasefree survival (DFS), longer overall survival (OS) or extended lifespan.Methods and systems of the invention are directed to molecular profilingof cancer on an individual basis that can provide alternatives fortreatment that may be convention or alternative to conventionaltreatment regimens. For example, alternative treatment regimes can beselected through molecular profiling methods of the invention where, adisease is refractory to current therapies, e.g., after a cancer hasdeveloped resistance to a standard-of-care treatment. Illustrativeschemes for using molecular profiling to identify a treatment regime areshown in FIGS. 2, 39 and 43, each of which is described in furtherdetail herein. Thus, molecular profiling provides a personalizedapproach to selecting candidate treatments that are likely to benefit acancer. In embodiments, the molecular profiling method is used toidentify therapies for patients with poor prognosis, such as those withmetastatic disease or those whose cancer has progressed on standardfront line therapies, or whose cancer has progressed on multiplechemotherapeutic or hormonal regimens.

Personalized medicine based on pharmacogenetic insights, such as thoseprovided by molecular profiling according to the invention, isincreasingly taken for granted by some practitioners and the lay press,but forms the basis of hope for improved cancer therapy. However,molecular profiling as taught herein represents a fundamental departurefrom the traditional approach to oncologic therapy where for the mostpart, patients are grouped together and treated with approaches that arebased on findings from light microscopy and disease stage.Traditionally, differential response to a particular therapeuticstrategy has only been determined after the treatment was given, i.e. aposteriori. The “standard” approach to disease treatment relies on whatis generally true about a given cancer diagnosis and treatment responsehas been vetted by randomized phase III clinical trials and forms the“standard of care” in medical practice. The results of these trials havebeen codified in consensus statements by guidelines organizations suchas the National Comprehensive Cancer Network and The American Society ofClinical Oncology. The NCCN Compendium™ contains authoritative,scientifically derived information designed to support decision-makingabout the appropriate use of drugs and biologics in patients withcancer. The NCCN Compendium™ is recognized by the Centers for Medicareand Medicaid Services (CMS) and United Healthcare as an authoritativereference for oncology coverage policy. On-compendium treatments arethose recommended by such guides. The biostatistical methods used tovalidate the results of clinical trials rely on minimizing differencesbetween patients, and are based on declaring the likelihood of errorthat one approach is better than another for a patient group definedonly by light microscopy and stage, not by individual differences intumors. The molecular profiling methods of the invention exploit suchindividual differences. The methods can provide candidate treatmentsthat can be then selected by a physician for treating a patient. In astudy of such an approach presented in Example 4 herein, the resultswere profound: in 66 consecutive patients, the treating oncologist nevermanaged to identify the molecular target selected by the test, and 27%of patients whose treatment was guided by molecular profiling managed aremission 1.3× longer than their previous best response. At present,such results are virtually unheard of result in the salvage therapysetting.

Molecular profiling can be used to provide a comprehensive view of thebiological state of a sample. In an embodiment, molecular profiling isused for whole tumor profiling. Accordingly, a number of molecularapproaches are used to assess the state of a tumor. The whole tumorprofiling can be used for selecting a candidate treatment for a tumor.Molecular profiling can be used to select candidate therapeutics on anysample for any stage of a disease. In embodiment, the methods of theinvention are used to profile a newly diagnosed cancer. The candidatetreatments indicated by the molecular profiling can be used to select atherapy for treating the newly diagnosed cancer. In other embodiments,the methods of the invention are used to profile a cancer that hasalready been treated, e.g., with one or more standard-of-care therapy.In embodiments, the cancer is refractory to the prior treatment/s. Forexample, the cancer may be refractory to the standard of care treatmentsfor the cancer. The cancer can be a metastatic cancer or other recurrentcancer. The treatments can be on-compendium or off-compendiumtreatments.

Molecular profiling can be performed by any known means for detecting amolecule in a biological sample. Molecular profiling comprises methodsthat include but are not limited to, nucleic acid sequencing, such as aDNA sequencing or mRNA sequencing; immunohistochemistry (IHC); in situhybridization (ISH); fluorescent in situ hybridization (FISH); varioustypes of microarray (mRNA expression arrays, low density arrays, proteinarrays, etc); various types of sequencing (Sanger, pyrosequencing, etc);comparative genomic hybridization (CGH); NextGen sequencing; Northernblot; Southern blot; immunoassay; and any other appropriate technique toassay the presence or quantity of a biological molecule of interest. Invarious embodiments of the invention, any one or more of these methodscan be used concurrently or subsequent to each other for assessingtarget genes disclosed herein.

Molecular profiling of individual samples is used to select one or morecandidate treatments for a disorder in a subject, e.g., by identifyingtargets for drugs that may be effective for a given cancer. For example,the candidate treatment can be a treatment known to have an effect oncells that differentially express genes as identified by molecularprofiling techniques, an experimental drug, a government or regulatoryapproved drug or any combination of such drugs, which may have beenstudied and approved for a particular indication that is the same as ordifferent from the indication of the subject from whom a biologicalsample is obtain and molecularly profiled.

When multiple biomarker targets are revealed by assessing target genesby molecular profiling, one or more decision rules can be put in placeto prioritize the selection of certain therapeutic agent for treatmentof an individual on a personalized basis. Rules of the invention aideprioritizing treatment, e.g., direct results of molecular profiling,anticipated efficacy of therapeutic agent, prior history with the sameor other treatments, expected side effects, availability of therapeuticagent, cost of therapeutic agent, drug-drug interactions, and otherfactors considered by a treating physician. Based on the recommended andprioritized therapeutic agent targets, a physician can decide on thecourse of treatment for a particular individual. Accordingly, molecularprofiling methods and systems of the invention can select candidatetreatments based on individual characteristics of diseased cells, e.g.,tumor cells, and other personalized factors in a subject in need oftreatment, as opposed to relying on a traditional one-size fits allapproach that is conventionally used to treat individuals suffering froma disease, especially cancer. In some cases, the recommended treatmentsare those not typically used to treat the disease or disorder inflictingthe subject. In some cases, the recommended treatments are used afterstandard-of-care therapies are no longer providing adequate efficacy.

The treating physician can use the results of the molecular profilingmethods to optimize a treatment regimen for a patient. The candidatetreatment identified by the methods of the invention can be used totreat a patient; however, such treatment is not required of the methods.Indeed, the analysis of molecular profiling results and identificationof candidate treatments based on those results can be automated and doesnot require physician involvement.

Biological Entities

Nucleic acids include deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, orcomplements thereof. Nucleic acids can contain known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleic acidsequence can encompass conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid can be used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence may implicitly encompass theparticular sequence and “splice variants” and nucleic acid sequencesencoding truncated forms. Similarly, a particular protein encoded by anucleic acid can encompass any protein encoded by a splice variant ortruncated form of that nucleic acid. “Splice variants,” as the namesuggests, are products of alternative splicing of a gene. Aftertranscription, an initial nucleic acid transcript may be spliced suchthat different (alternate) nucleic acid splice products encode differentpolypeptides. Mechanisms for the production of splice variants vary, butinclude alternate splicing of exons. Alternate polypeptides derived fromthe same nucleic acid by read-through transcription are also encompassedby this definition. Any products of a splicing reaction, includingrecombinant forms of the splice products, are included in thisdefinition. Nucleic acids can be truncated at the 5′ end or at the 3′end. Polypeptides can be truncated at the N-terminal end or theC-terminal end. Truncated versions of nucleic acid or polypeptidesequences can be naturally occurring or created using recombinanttechniques.

The terms “genetic variant” and “nucleotide variant” are used hereininterchangeably to refer to changes or alterations to the referencehuman gene or cDNA sequence at a particular locus, including, but notlimited to, nucleotide base deletions, insertions, inversions, andsubstitutions in the coding and non-coding regions. Deletions may be ofa single nucleotide base, a portion or a region of the nucleotidesequence of the gene, or of the entire gene sequence. Insertions may beof one or more nucleotide bases. The genetic variant or nucleotidevariant may occur in transcriptional regulatory regions, untranslatedregions of mRNA, exons, introns, exon/intron junctions, etc. The geneticvariant or nucleotide variant can potentially result in stop codons,frame shifts, deletions of amino acids, altered gene transcript spliceforms or altered amino acid sequence.

An allele or gene allele comprises generally a naturally occurring genehaving a reference sequence or a gene containing a specific nucleotidevariant.

A haplotype refers to a combination of genetic (nucleotide) variants ina region of an mRNA or a genomic DNA on a chromosome found in anindividual. Thus, a haplotype includes a number of genetically linkedpolymorphic variants which are typically inherited together as a unit.

As used herein, the term “amino acid variant” is used to refer to anamino acid change to a reference human protein sequence resulting fromgenetic variants or nucleotide variants to the reference human geneencoding the reference protein. The term “amino acid variant” isintended to encompass not only single amino acid substitutions, but alsoamino acid deletions, insertions, and other significant changes of aminoacid sequence in the reference protein.

The term “genotype” as used herein means the nucleotide characters at aparticular nucleotide variant marker (or locus) in either one allele orboth alleles of a gene (or a particular chromosome region). With respectto a particular nucleotide position of a gene of interest, thenucleotide(s) at that locus or equivalent thereof in one or both allelesform the genotype of the gene at that locus. A genotype can behomozygous or heterozygous. Accordingly, “genotyping” means determiningthe genotype, that is, the nucleotide(s) at a particular gene locus.Genotyping can also be done by determining the amino acid variant at aparticular position of a protein which can be used to deduce thecorresponding nucleotide variant(s).

The term “locus” refers to a specific position or site in a genesequence or protein. Thus, there may be one or more contiguousnucleotides in a particular gene locus, or one or more amino acids at aparticular locus in a polypeptide. Moreover, a locus may refer to aparticular position in a gene where one or more nucleotides have beendeleted, inserted, or inverted.

Unless specified otherwise or understood by one of skill in art, theterms “polypeptide,” “protein,” and “peptide” are used hereininterchangeably to refer to an amino acid chain in which the amino acidresidues are linked by covalent peptide bonds. The amino acid chain canbe of any length of at least two amino acids, including full-lengthproteins. Unless otherwise specified, polypeptide, protein, and peptidealso encompass various modified forms thereof, including but not limitedto glycosylated forms, phosphorylated forms, etc. A polypeptide, proteinor peptide can also be referred to as a gene product.

Lists of gene and gene products that can be assayed by molecularprofiling techniques are presented herein. Lists of genes may bepresented in the context of molecular profiling techniques that detect agene product (e.g., an mRNA or protein). One of skill will understandthat this implies detection of the gene product of the listed genes.Similarly, lists of gene products may be presented in the context ofmolecular profiling techniques that detect a gene sequence or copynumber. One of skill will understand that this implies detection of thegene corresponding to the gene products, including as an example DNAencoding the gene products. As will be appreciated by those skilled inthe art, a “biomarker” or “marker” comprises a gene and/or gene productdepending on the context.

The terms “label” and “detectable label” can refer to any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, chemical or similar methods. Such labels includebiotin for staining with labeled streptavidin conjugate, magnetic beads(e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein, Texas red,rhodamine, green fluorescent protein, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcalorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc) beads. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means ofdetecting such labels are well known to those of skill in the art. Thus,for example, radiolabels may be detected using photographic film orscintillation counters, fluorescent markers may be detected using aphotodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,and calorimetric labels are detected by simply visualizing the coloredlabel. Labels can include, e.g., ligands that bind to labeledantibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled ligand. An introduction to labels, labeling procedures anddetection of labels is found in Polak and Van Noorden Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and inHaugland Handbook of Fluorescent Probes and Research Chemicals, acombined handbook and catalogue Published by Molecular Probes, Inc.(1996).

Detectable labels include, but are not limited to, nucleotides (labeledor unlabelled), compomers, sugars, peptides, proteins, antibodies,chemical compounds, conducting polymers, binding moieties such asbiotin, mass tags, calorimetric agents, light emitting agents,chemiluminescent agents, light scattering agents, fluorescent tags,radioactive tags, charge tags (electrical or magnetic charge), volatiletags and hydrophobic tags, biomolecules (e.g., members of a binding pairantibody/antigen, antibody/antibody, antibody/antibody fragment,antibody/antibody receptor, antibody/protein A or protein G,hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folicacid/folate binding protein, vitamin B12/intrinsic factor, chemicalreactive group/complementary chemical reactive group (e.g.,sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative,amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonylhalides) and the like.

The term “antibody” as used herein encompasses naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof, (e.g., Fab′,F(ab′)₂, Fab, Fv and rIgG). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.). See also, e.g., Kuby,J., Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York (1998). Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art. See, e.g., Winter and Harris, Immunol Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications,New York, 1988; Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrebaeck, Antibody Engineering, 2d ed.(Oxford University Press 1995); each of which is incorporated herein byreference.

Unless otherwise specified, antibodies can include both polyclonal andmonoclonal antibodies. Antibodies also include genetically engineeredforms such as chimeric antibodies (e.g., humanized murine antibodies)and heteroconjugate antibodies (e.g., bispecific antibodies). The termalso refers to recombinant single chain Fv fragments (scFv). The termantibody also includes bivalent or bispecific molecules, diabodies,triabodies, and tetrabodies. Bivalent and bispecific molecules aredescribed in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack andPluckthun (1992) Biochemistry 31:1579, Holliger et al. (1993) Proc NatlAcad Sci USA. 90:6444, Gruber et al. (1994) J Immunol: 5368, Zhu et al.(1997) Protein Sci 6:781, Hu et al. (1997) Cancer Res. 56:3055, Adams etal. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) ProteinEng. 8:301.

Typically, an antibody has a heavy and light chain. Each heavy and lightchain contains a constant region and a variable region, (the regions arealso known as “domains”). Light and heavy chain variable regions containfour framework regions interrupted by three hyper-variable regions, alsocalled complementarity-determining regions (CDRs). The extent of theframework regions and CDRs have been defined. The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three dimensionalspaces. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. References to V_(H) refer to the variable regionof an immunoglobulin heavy chain of an antibody, including the heavychain of an Fv, scFv, or Fab. References to V_(L) refer to the variableregion of an immunoglobulin light chain, including the light chain of anFv, scFv, dsFv or Fab.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site. A “chimericantibody” is an immunoglobulin molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

A “humanized antibody” is an immunoglobulin molecule that containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

The terms “epitope” and “antigenic determinant” refer to a site on anantigen to which an antibody binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

The terms “primer”, “probe,” and “oligonucleotide” are used hereininterchangeably to refer to a relatively short nucleic acid fragment orsequence. They can comprise DNA, RNA, or a hybrid thereof, or chemicallymodified analog or derivatives thereof. Typically, they aresingle-stranded. However, they can also be double-stranded having twocomplementing strands which can be separated by denaturation. Normally,primers, probes and oligonucleotides have a length of from about 8nucleotides to about 200 nucleotides, preferably from about 12nucleotides to about 100 nucleotides, and more preferably about 18 toabout 50 nucleotides. They can be labeled with detectable markers ormodified using conventional manners for various molecular biologicalapplications.

The term “isolated” when used in reference to nucleic acids (e.g.,genomic DNAs, cDNAs, mRNAs, or fragments thereof) is intended to meanthat a nucleic acid molecule is present in a form that is substantiallyseparated from other naturally occurring nucleic acids that are normallyassociated with the molecule. Because a naturally existing chromosome(or a viral equivalent thereof) includes a long nucleic acid sequence,an isolated nucleic acid can be a nucleic acid molecule having only aportion of the nucleic acid sequence in the chromosome but not one ormore other portions present on the same chromosome. More specifically,an isolated nucleic acid can include naturally occurring nucleic acidsequences that flank the nucleic acid in the naturally existingchromosome (or a viral equivalent thereof). An isolated nucleic acid canbe substantially separated from other naturally occurring nucleic acidsthat are on a different chromosome of the same organism. An isolatednucleic acid can also be a composition in which the specified nucleicacid molecule is significantly enriched so as to constitute at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of thetotal nucleic acids in the composition.

An isolated nucleic acid can be a hybrid nucleic acid having thespecified nucleic acid molecule covalently linked to one or more nucleicacid molecules that are not the nucleic acids naturally flanking thespecified nucleic acid. For example, an isolated nucleic acid can be ina vector. In addition, the specified nucleic acid may have a nucleotidesequence that is identical to a naturally occurring nucleic acid or amodified form or mutein thereof having one or more mutations such asnucleotide substitution, deletion/insertion, inversion, and the like.

An isolated nucleic acid can be prepared from a recombinant host cell(in which the nucleic acids have been recombinantly amplified and/orexpressed), or can be a chemically synthesized nucleic acid having anaturally occurring nucleotide sequence or an artificially modified formthereof.

The term “isolated polypeptide” as used herein is defined as apolypeptide molecule that is present in a form other than that found innature. Thus, an isolated polypeptide can be a non-naturally occurringpolypeptide. For example, an isolated polypeptide can be a “hybridpolypeptide.” An isolated polypeptide can also be a polypeptide derivedfrom a naturally occurring polypeptide by additions or deletions orsubstitutions of amino acids. An isolated polypeptide can also be a“purified polypeptide” which is used herein to mean a composition orpreparation in which the specified polypeptide molecule is significantlyenriched so as to constitute at least 10% of the total protein contentin the composition. A “purified polypeptide” can be obtained fromnatural or recombinant host cells by standard purification techniques,or by chemically synthesis, as will be apparent to skilled artisans.

The terms “hybrid protein,” “hybrid polypeptide,” “hybrid peptide,”“fusion protein,” “fusion polypeptide,” and “fusion peptide” are usedherein interchangeably to mean a non-naturally occurring polypeptide orisolated polypeptide having a specified polypeptide molecule covalentlylinked to one or more other polypeptide molecules that do not link tothe specified polypeptide in nature. Thus, a “hybrid protein” may be twonaturally occurring proteins or fragments thereof linked together by acovalent linkage. A “hybrid protein” may also be a protein formed bycovalently linking two artificial polypeptides together. Typically butnot necessarily, the two or more polypeptide molecules are linked or“fused” together by a peptide bond forming a single non-branchedpolypeptide chain.

The term “high stringency hybridization conditions,” when used inconnection with nucleic acid hybridization, includes hybridizationconducted overnight at 42° C. in a solution containing 50% formamide,5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH7.6, 5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured and sheared salmon sperm DNA, with hybridization filterswashed in 0.1×SSC at about 65° C. The term “moderate stringenthybridization conditions,” when used in connection with nucleic acidhybridization, includes hybridization conducted overnight at 37° C. in asolution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate, pH 7.6, 5×Denhardt's solution, 10%dextran sulfate, and 20 microgram/ml denatured and sheared salmon spermDNA, with hybridization filters washed in 1×SSC at about 50° C. It isnoted that many other hybridization methods, solutions and temperaturescan be used to achieve comparable stringent hybridization conditions aswill be apparent to skilled artisans.

For the purpose of comparing two different nucleic acid or polypeptidesequences, one sequence (test sequence) may be described to be aspecific percentage identical to another sequence (comparison sequence).The percentage identity can be determined by the algorithm of Karlin andAltschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which isincorporated into various BLAST programs. The percentage identity can bedetermined by the “BLAST 2 Sequences” tool, which is available at theNational Center for Biotechnology Information (NCBI) website. SeeTatusova and Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999). Forpairwise DNA-DNA comparison, the BLASTN program is used with defaultparameters (e.g., Match: 1; Mismatch: −2; Open gap: 5 penalties;extension gap: 2 penalties; gap x_dropoff: 50; expect: 10; and wordsize: 11, with filter). For pairwise protein-protein sequencecomparison, the BLASTP program can be employed using default parameters(e.g., Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15;expect: 10.0; and wordsize: 3, with filter). Percent identity of twosequences is calculated by aligning a test sequence with a comparisonsequence using BLAST, determining the number of amino acids ornucleotides in the aligned test sequence that are identical to aminoacids or nucleotides in the same position of the comparison sequence,and dividing the number of identical amino acids or nucleotides by thenumber of amino acids or nucleotides in the comparison sequence. WhenBLAST is used to compare two sequences, it aligns the sequences andyields the percent identity over defined, aligned regions. If the twosequences are aligned across their entire length, the percent identityyielded by the BLAST is the percent identity of the two sequences. IfBLAST does not align the two sequences over their entire length, thenthe number of identical amino acids or nucleotides in the unalignedregions of the test sequence and comparison sequence is considered to bezero and the percent identity is calculated by adding the number ofidentical amino acids or nucleotides in the aligned regions and dividingthat number by the length of the comparison sequence. Various versionsof the BLAST programs can be used to compare sequences, e.g., BLAST2.1.2 or BLAST+ 2.2.22.

A subject or individual can be any animal which may benefit from themethods of the invention, including, e.g., humans and non-human mammals,such as primates, rodents, horses, dogs and cats. Subjects includewithout limitation a eukaryotic organisms, most preferably a mammal suchas a primate, e.g., chimpanzee or human, cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. Subjectsspecifically intended for treatment using the methods described hereininclude humans. A subject may be referred to as an individual or apatient.

Treatment of a disease or individual according to the invention is anapproach for obtaining beneficial or desired medical results, includingclinical results, but not necessarily a cure. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation or amelioration of one or more symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, preventing spread of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment or if receiving a differenttreatment. A treatment can include administration of a therapeuticagent, which can be an agent that exerts a cytotoxic, cytostatic, orimmunomodulatory effect on diseased cells, e.g., cancer cells, or othercells that may promote a diseased state, e.g., activated immune cells.Therapeutic agents selected by the methods of the invention are notlimited. Any therapeutic agent can be selected where a link can be madebetween molecular profiling and potential efficacy of the agent.Therapeutic agents include without limitation drugs, pharmaceuticals,small molecules, protein therapies, antibody therapies, viral therapies,gene therapies, and the like. Cancer treatments or therapies includeapoptosis-mediated and non-apoptosis mediated cancer therapiesincluding, without limitation, chemotherapy, hormonal therapy,radiotherapy, immunotherapy, and combinations thereof. Chemotherapeuticagents comprise therapeutic agents and combinations of therapeuticagents that treat, cancer cells, e.g., by killing those cells. Examplesof different types of chemotherapeutic drugs include without limitationalkylating agents (e.g., nitrogen mustard derivatives, ethylenimines,alkylsulfonates, hydrazines and triazines, nitrosureas, and metalsalts), plant alkaloids (e.g., vinca alkaloids, taxanes,podophyllotoxins, and camptothecan analogs), antitumor antibiotics(e.g., anthracyclines, chromomycins, and the like), antimetabolites(e.g., folic acid antagonists, pyrimidine antagonists, purineantagonists, and adenosine deaminase inhibitors), topoisomerase Iinhibitors, topoisomerase II inhibitors, and miscellaneousantineoplastics (e.g., ribonucleotide reductase inhibitors,adrenocortical steroid inhibitors, enzymes, antimicrotubule agents, andretinoids).

A biomarker refers generally to a molecule, including without limitationa gene or product thereof, nucleic acids (e.g., DNA, RNA),protein/peptide/polypeptide, carbohydrate structure, lipid, glycolipid,characteristics of which can be detected in a tissue or cell to provideinformation that is predictive, diagnostic, prognostic and/ortheranostic for sensitivity or resistance to candidate treatment.

Biological Samples

A sample as used herein includes any relevant biological sample that canbe used for molecular profiling, e.g., sections of tissues such asbiopsy or tissue removed during surgical or other procedures, bodilyfluids, autopsy samples, and frozen sections taken for histologicalpurposes. Such samples include blood and blood fractions or products(e.g., serum, buffy coat, plasma, platelets, red blood cells, and thelike), sputum, malignant effusion, cheek cells tissue, cultured cells(e.g., primary cultures, explants, and transformed cells), stool, urine,other biological or bodily fluids (e.g., prostatic fluid, gastric fluid,intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, and thelike), etc. The sample can comprise biological material that is a freshfrozen & formalin fixed paraffin embedded (FFPE) block, formalin-fixedparaffin embedded, or is within an RNA preservative+formalin fixative.More that one sample of more than one type can be used for each patient.

The sample used in the methods described herein can be a formalin fixedparaffin embedded (FFPE) sample. The FFPE sample can be one or more offixed tissue, unstained slides, bone marrow core or clot, core needlebiopsy, malignant fluids and fine needle aspirate (FNA). In anembodiment, the fixed tissue comprises a tumor containing formalin fixedparaffin embedded (FFPE) block from a surgery or biopsy. In anotherembodiment, the unstained slides comprise unstained, charged, unbakedslides from a paraffin block. In another embodiment, bone marrow core orclot comprises a decalcified core. A formalin fixed core and/or clot canbe paraffin-embedded. In still another embodiment, the core needlebiopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 3-4,paraffin embedded biopsy samples. An 18 gauge needle biopsy can be used.The malignant fluid can comprise a sufficient volume of freshpleural/ascitic fluid to produce a 5×5×2 mm cell pellet. The fluid canbe formalin fixed in a paraffin block. In an embodiment, the core needlebiopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 4-6,paraffin embedded aspirates.

A sample may be processed according to techniques understood by those inthe art. A sample can be without limitation fresh, frozen or fixed cellsor tissue. In some embodiments, a sample comprises formalin-fixedparaffin-embedded (FFPE) tissue, fresh tissue or fresh frozen (FF)tissue. A sample can comprise cultured cells, including primary orimmortalized cell lines derived from a subject sample. A sample can alsorefer to an extract from a sample from a subject. For example, a samplecan comprise DNA, RNA or protein extracted from a tissue or a bodilyfluid. Many techniques and commercial kits are available for suchpurposes. The fresh sample from the individual can be treated with anagent to preserve RNA prior to further processing, e.g., cell lysis andextraction. Samples can include frozen samples collected for otherpurposes. Samples can be associated with relevant information such asage, gender, and clinical symptoms present in the subject; source of thesample; and methods of collection and storage of the sample. A sample istypically obtained from a subject.

A biopsy comprises the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itself.Any biopsy technique known in the art can be applied to the molecularprofiling methods of the present invention. The biopsy technique appliedcan depend on the tissue type to be evaluated (e.g., colon, prostate,kidney, bladder, lymph node, liver, bone marrow, blood cell, lung,breast, etc.), the size and type of the tumor (e.g., solid or suspended,blood or ascites), among other factors. Representative biopsy techniquesinclude, but are not limited to, excisional biopsy, incisional biopsy,needle biopsy, surgical biopsy, and bone marrow biopsy. An “excisionalbiopsy” refers to the removal of an entire tumor mass with a smallmargin of normal tissue surrounding it. An “incisional biopsy” refers tothe removal of a wedge of tissue that includes a cross-sectionaldiameter of the tumor. Molecular profiling can use a “core-needlebiopsy” of the tumor mass, or a “fine-needle aspiration biopsy” whichgenerally obtains a suspension of cells from within the tumor mass.Biopsy techniques are discussed, for example, in Harrison's Principlesof Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70,and throughout Part V.

Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) can becarried out generally as in PCR Protocols: A Guide to Methods andApplications, Academic Press, San Diego, Calif. (1990).

Vesicles

The sample can comprise vesicles. Methods of the invention can includeassessing one or more vesicles, including assessing vesicle populations.A vesicle, as used herein, is a membrane vesicle that is shed fromcells. Vesicles or membrane vesicles include without limitation:circulating microvesicles (cMVs), microvesicle, exosome, nanovesicle,dexosome, bleb, blebby, prostasome, microparticle, intralumenal vesicle,membrane fragment, intralumenal endosomal vesicle, endosomal-likevesicle, exocytosis vehicle, endosome vesicle, endosomal vesicle,apoptotic body, multivesicular body, secretory vesicle, phospholipidvesicle, liposomal vesicle, argosome, texasome, secresome, tolerosome,melanosome, oncosome, or exocytosed vehicle. Furthermore, althoughvesicles may be produced by different cellular processes, the methods ofthe invention are not limited to or reliant on any one mechanism,insofar as such vesicles are present in a biological sample and arecapable of being characterized by the methods disclosed herein. Unlessotherwise specified, methods that make use of a species of vesicle canbe applied to other types of vesicles. Vesicles comprise sphericalstructures with a lipid bilayer similar to cell membranes whichsurrounds an inner compartment which can contain soluble components,sometimes referred to as the payload. In some embodiments, the methodsof the invention make use of exosomes, which are small secreted vesiclesof about 40-100 nm in diameter. For a review of membrane vesicles,including types and characterizations, see Thery et al., Nat RevImmunol. 2009 August; 9(8):581-93. Some properties of different types ofvesicles include those in Table 1:

TABLE 1 Vesicle Properties Exosome- Membrane like Apoptotic FeatureExosomes Microvesicles Ectosomes particles vesicles vesicles Size 50-100nm 100-1,000 nm 50-200 nm 50-80 nm 20-50 nm 50-500 nm Density in1.13-1.19 g/ml 1.04-1.07 g/ml 1.1 g/ml 1.16-1.28 g/ml sucrose EM Cupshape Irregular Bilamellar Round Irregular Heterogeneous appearanceshape, round shape electron structures dense Sedimentation 100,000 g10,000 g 160,000-200,000 g 100,000-200,000 g 175,000 g 1,200 g, 10,000g, 100,000 g Lipid Enriched in Expose PPS Enriched in No lipidcomposition cholesterol, cholesterol rafts sphingomyelin and andceramide; diacylglycerol; contains lipid expose PPS rafts; expose PPSMajor Tetraspanins Integrins, CR1 and CD133; no TNFRI Histones protein(e.g., CD63, selectins and proteolytic CD63 markers CD9), Alix, CD40ligand enzymes; no TSG101 CD63 Intracellular Internal Plasma PlasmaPlasma origin compartments membrane membrane membrane (endosomes)Abbreviations: phosphatidylserine (PPS); electron microscopy (EM)

Vesicles include shed membrane bound particles, or “microparticles,”that are derived from either the plasma membrane or an internalmembrane. Vesicles can be released into the extracellular environmentfrom cells. Cells releasing vesicles include without limitation cellsthat originate from, or are derived from, the ectoderm, endoderm, ormesoderm. The cells may have undergone genetic, environmental, and/orany other variations or alterations. For example, the cell can be tumorcells. A vesicle can reflect any changes in the source cell, and therebyreflect changes in the originating cells, e.g., cells having variousgenetic mutations. In one mechanism, a vesicle is generatedintracellularly when a segment of the cell membrane spontaneouslyinvaginates and is ultimately exocytosed (see for example, Keller etal., Immunol. Lett. 107 (2): 102-8 (2006)). Vesicles also includecell-derived structures bounded by a lipid bilayer membrane arising fromboth herniated evagination (blebbing) separation and sealing of portionsof the plasma membrane or from the export of any intracellularmembrane-bounded vesicular structure containing variousmembrane-associated proteins of tumor origin, including surface-boundmolecules derived from the host circulation that bind selectively to thetumor-derived proteins together with molecules contained in the vesiclelumen, including but not limited to tumor-derived microRNAs orintracellular proteins. Blebs and blebbing are further described inCharras et al., Nature Reviews Molecular and Cell Biology, Vol. 9, No.11, p. 730-736 (2008). A vesicle shed into circulation or bodily fluidsfrom tumor cells may be referred to as a “circulating tumor-derivedvesicle.” When such vesicle is an exosome, it may be referred to as acirculating-tumor derived exosome (CTE). In some instances, a vesiclecan be derived from a specific cell of origin. CTE, as with acell-of-origin specific vesicle, typically have one or more uniquebiomarkers that permit isolation of the CTE or cell-of-origin specificvesicle, e.g., from a bodily fluid and sometimes in a specific manner.For example, a cell or tissue specific markers are used to identify thecell of origin. Examples of such cell or tissue specific markers aredisclosed herein and can further be accessed in the Tissue-specific GeneExpression and Regulation (TiGER) Database, available atbioinfo.wilmer.jhu.edu/tiger/; Liu et al. (2008) TiGER a database fortissue-specific gene expression and regulation. BMC Bioinformatics.9:271; TissueDistributionDBs, available atgenome.dkfz-heidelberg.de/menu/tissue_db/index.html.

A vesicle can have a diameter of greater than about 10 nm, 20 nm, or 30nm. A vesicle can have a diameter of greater than 40 nm, 50 nm, 100 nm,200 nm, 500 nm, 1000 nm or greater than 10,000 nm. A vesicle can have adiameter of about 30-1000 nm, about 30-800 nm, about 30-200 nm, or about30-100 nm. In some embodiments, the vesicle has a diameter of less than10,000 nm, 1000 nm, 800 nm, 500 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm,20 nm or less than 10 nm. As used herein the term “about” in referenceto a numerical value means that variations of 10% above or below thenumerical value are within the range ascribed to the specified value.Typical sizes for various types of vesicles are shown in Table 1.Vesicles can be assessed to measure the diameter of a single vesicle orany number of vesicles. For example, the range of diameters of a vesiclepopulation or an average diameter of a vesicle population can bedetermined. Vesicle diameter can be assessed using methods known in theart, e.g., imaging technologies such as electron microscopy. In anembodiment, a diameter of one or more vesicles is determined usingoptical particle detection. See, e.g., U.S. Pat. No. 7,751,053, entitled“Optical Detection and Analysis of Particles” and issued Jul. 6, 2010;and U.S. Pat. No. 7,399,600, entitled “Optical Detection and Analysis ofParticles” and issued Jul. 15, 2010.

In some embodiments, vesicles are directly assayed from a biologicalsample without prior isolation, purification, or concentration from thebiological sample. For example, the amount of vesicles in the sample canby itself provide a biosignature that provides a diagnostic, prognosticor theranostic determination. Alternatively, the vesicle in the samplemay be isolated, captured, purified, or concentrated from a sample priorto analysis. As noted, isolation, capture or purification as used hereincomprises partial isolation, partial capture or partial purificationapart from other components in the sample. Vesicle isolation can beperformed using various techniques as described herein or known in theart, including without limitation size exclusion chromatography, densitygradient centrifugation, differential centrifugation, nanomembraneultrafiltration, immunoabsorbent capture, affinity purification,affinity capture, immunoassay, immunoprecipitation, microfluidicseparation, flow cytometry or combinations thereof.

Vesicles can be assessed to provide a phenotypic characterization bycomparing vesicle characteristics to a reference. In some embodiments,surface antigens on a vesicle are assessed. A vesicle or vesiclepopulation carrying a specific marker can be referred to as a positive(biomarker+) vesicle or vesicle population. For example, a DLL4+population refers to a vesicle population associated with DLL4.Conversely, a DLL4− population would not be associated with DLL4. Thesurface antigens can provide an indication of the anatomical originand/or cellular of the vesicles and other phenotypic information, e.g.,tumor status. For example, vesicles found in a patient sample can beassessed for surface antigens indicative of colorectal origin and thepresence of cancer, thereby identifying vesicles associated withcolorectal cancer cells. The surface antigens may comprise anyinformative biological entity that can be detected on the vesiclemembrane surface, including without limitation surface proteins, lipids,carbohydrates, and other membrane components. For example, positivedetection of colon derived vesicles expressing tumor antigens canindicate that the patient has colorectal cancer. As such, methods of theinvention can be used to characterize any disease or conditionassociated with an anatomical or cellular origin, by assessing, forexample, disease-specific and cell-specific biomarkers of one or morevesicles obtained from a subject.

In embodiments, one or more vesicle payloads are assessed to provide aphenotypic characterization. The payload with a vesicle comprises anyinformative biological entity that can be detected as encapsulatedwithin the vesicle, including without limitation proteins and nucleicacids, e.g., genomic or cDNA, mRNA, or functional fragments thereof, aswell as microRNAs (miRs). In addition, methods of the invention aredirected to detecting vesicle surface antigens (in addition or exclusiveto vesicle payload) to provide a phenotypic characterization. Forexample, vesicles can be characterized by using binding agents (e.g.,antibodies or aptamers) that are specific to vesicle surface antigens,and the bound vesicles can be further assessed to identify one or morepayload components disclosed therein. As described herein, the levels ofvesicles with surface antigens of interest or with payload of interestcan be compared to a reference to characterize a phenotype. For example,overexpression in a sample of cancer-related surface antigens or vesiclepayload, e.g., a tumor associated mRNA or microRNA, as compared to areference, can indicate the presence of cancer in the sample. Thebiomarkers assessed can be present or absent, increased or reduced basedon the selection of the desired target sample and comparison of thetarget sample to the desired reference sample. Non-limiting examples oftarget samples include: disease; treated/not-treated; different timepoints, such as a in a longitudinal study; and non-limiting examples ofreference sample: non-disease; normal; different time points; andsensitive or resistant to candidate treatment(s). In an embodiment,molecular profiling of the invention comprises analysis ofmicrovesicles, such as circulating microvesicles.

MicroRNA

Various biomarker molecules can be assessed in biological samples orvesicles obtained from such biological samples. MicroRNAs comprise oneclass biomarkers assessed via methods of the invention. MicroRNAs, alsoreferred to herein as miRNAs or miRs, are short RNA strandsapproximately 21-23 nucleotides in length. MiRNAs are encoded by genesthat are transcribed from DNA but are not translated into protein andthus comprise non-coding RNA. The miRs are processed from primarytranscripts known as pri-miRNA to short stem-loop structures calledpre-miRNA and finally to the resulting single strand miRNA. Thepre-miRNA typically forms a structure that folds back on itself inself-complementary regions. These structures are then processed by thenuclease Dicer in animals or DCL1 in plants. Mature miRNA molecules arepartially complementary to one or more messenger RNA (mRNA) moleculesand can function to regulate translation of proteins. Identifiedsequences of miRNA can be accessed at publicly available databases, suchas www.microRNA.org, www.mirbase.org, orwww.mirz.unibas.ch/cgi/miRNA.cgi.

miRNAs are generally assigned a number according to the namingconvention “mir-[number].” The number of a miRNA is assigned accordingto its order of discovery relative to previously identified miRNAspecies. For example, if the last published miRNA was mir-121, the nextdiscovered miRNA will be named mir-122, etc. When a miRNA is discoveredthat is homologous to a known miRNA from a different organism, the namecan be given an optional organism identifier, of the form [organismidentifier]-mir-[number]. Identifiers include hsa for Homo sapiens andmmu for Mus Musculus. For example, a human homolog to mir-121 might bereferred to as hsa-mir-121 whereas the mouse homolog can be referred toas mmu-mir-121.

Mature microRNA is commonly designated with the prefix “miR” whereas thegene or precursor miRNA is designated with the prefix “mir.” Forexample, mir-121 is a precursor for miR-121. When differing miRNA genesor precursors are processed into identical mature miRNAs, thegenes/precursors can be delineated by a numbered suffix. For example,mir-121-1 and mir-121-2 can refer to distinct genes or precursors thatare processed into miR-121. Lettered suffixes are used to indicateclosely related mature sequences. For example, mir-121a and mir-121b canbe processed to closely related miRNAs miR-121a and miR-121b,respectively. In the context of the invention, any microRNA (miRNA ormiR) designated herein with the prefix mir-* or miR-* is understood toencompass both the precursor and/or mature species, unless otherwiseexplicitly stated otherwise.

Sometimes it is observed that two mature miRNA sequences originate fromthe same precursor. When one of the sequences is more abundant that theother, a “*” suffix can be used to designate the less common variant.For example, miR-121 would be the predominant product whereas miR-121*is the less common variant found on the opposite arm of the precursor.If the predominant variant is not identified, the miRs can bedistinguished by the suffix “5p” for the variant from the 5′ arm of theprecursor and the suffix “3p” for the variant from the 3′ arm. Forexample, miR-121-5p originates from the 5′ arm of the precursor whereasmiR-121-3p originates from the 3′ arm. Less commonly, the 5p and 3pvariants are referred to as the sense (“s”) and anti-sense (“as”) forms,respectively. For example, miR-121-5p may be referred to as miR-121-swhereas miR-121-3p may be referred to as miR-121-as.

The above naming conventions have evolved over time and are generalguidelines rather than absolute rules. For example, the let- andlin-families of miRNAs continue to be referred to by these monikers. Themir/miR convention for precursor/mature forms is also a guideline andcontext should be taken into account to determine which form is referredto. Further details of miR naming can be found at www.mirbase.org orAmbros et al., A uniform system for microRNA annotation, RNA 9:277-279(2003).

Plant miRNAs follow a different naming convention as described in Meyerset al., Plant Cell. 2008 20(12):3186-3190.

A number of miRNAs are involved in gene regulation, and miRNAs are partof a growing class of non-coding RNAs that is now recognized as a majortier of gene control. In some cases, miRNAs can interrupt translation bybinding to regulatory sites embedded in the 3′-UTRs of their targetmRNAs, leading to the repression of translation. Target recognitioninvolves complementary base pairing of the target site with the miRNA'sseed region (positions 2-8 at the miRNA's 5′ end), although the exactextent of seed complementarity is not precisely determined and can bemodified by 3′ pairing. In other cases, miRNAs function like smallinterfering RNAs (siRNA) and bind to perfectly complementary mRNAsequences to destroy the target transcript.

Characterization of a number of miRNAs indicates that they influence avariety of processes, including early development, cell proliferationand cell death, apoptosis and fat metabolism. For example, some miRNAs,such as lin-4, let-7, mir-14, mir-23, and bantam, have been shown toplay critical roles in cell differentiation and tissue development.Others are believed to have similarly important roles because of theirdifferential spatial and temporal expression patterns.

The miRNA database available at miRBase (www.mirbase.org) comprises asearchable database of published miRNA sequences and annotation. Furtherinformation about miRBase can be found in the following articles, eachof which is incorporated by reference in its entirety herein:Griffiths-Jones et al., miRBase: tools for microRNA genomics. NAR 200836(Database Issue):D154-D158; Griffiths-Jones et al., miRBase: microRNAsequences, targets and gene nomenclature. NAR 2006 34(DatabaseIssue):D140-D144; and Griffiths-Jones, S. The microRNA Registry. NAR2004 32 (Database Issue):D109-D111. Representative miRNAs contained inRelease 16 of miRBase, made available September 2010.

As described herein, microRNAs are known to be involved in cancer andother diseases and can be assessed in order to characterize a phenotypein a sample. See, e.g., Ferracin et al., Micromarkers: miRNAs in cancerdiagnosis and prognosis, Exp Rev Mol Diag, April 2010, Vol. 10, No. 3,Pages 297-308; Fabbri, miRNAs as molecular biomarkers of cancer, Exp RevMol Diag, May 2010, Vol. 10, No. 4, Pages 435-444. In an embodiment,molecular profiling of the invention comprises analysis of microRNA.

Techniques to isolate and characterize vesicles and miRs are known tothose of skill in the art. In addition to the methodology presentedherein, additional methods can be found in U.S. Pat. No. 7,888,035,entitled “METHODS FOR ASSESSING RNA PATTERNS” and issued Feb. 15, 2011;and U.S. Pat. No. 7,897,356, entitled “METHODS AND SYSTEMS OF USINGEXOSOMES FOR DETERMINING PHENOTYPES” and issued Mar. 1, 2011; andInternational Patent Publication Nos. WO/2011/066589, entitled “METHODSAND SYSTEMS FOR ISOLATING, STORING, AND ANALYZING VESICLES” and filedNov. 30, 2010; WO/2011/088226, entitled “DETECTION OF GASTROINTESTINALDISORDERS” and filed Jan. 13, 2011; WO/2011/109440, entitled “BIOMARKERSFOR THERANOSTICS” and filed Mar. 1, 2011; and WO/2011/127219, entitled“CIRCULATING BIOMARKERS FOR DISEASE” and filed Apr. 6, 2011, each ofwhich applications are incorporated by reference herein in theirentirety.

Circulating Biomarkers

Circulating biomarkers include biomarkers that are detectable in bodyfluids, such as blood, plasma, serum. Examples of circulating cancerbiomarkers include cardiac troponin T (cTnT), prostate specific antigen(PSA) for prostate cancer and CA125 for ovarian cancer. Circulatingbiomarkers according to the invention include any appropriate biomarkerthat can be detected in bodily fluid, including without limitationprotein, nucleic acids, e.g., DNA, mRNA and microRNA, lipids,carbohydrates and metabolites. Circulating biomarkers can includebiomarkers that are not associated with cells, such as biomarkers thatare membrane associated, embedded in membrane fragments, part of abiological complex, or free in solution. In one embodiment, circulatingbiomarkers are biomarkers that are associated with one or more vesiclespresent in the biological fluid of a subject. Circulating biomarkershave been identified for use in characterization of various phenotypes,such as detection of a cancer. See, e.g., Ahmed N, et al.,Proteomic-based identification of haptoglobin-1 precursor as a novelcirculating biomarker of ovarian cancer. Br. J. Cancer 2004; Mathelin etal., Circulating proteinic biomarkers and breast cancer, Gynecol Obstet.Fertil. 2006 July-August; 34(7-8):638-46. Epub 2006 Jul. 28; Ye et al.,Recent technical strategies to identify diagnostic biomarkers forovarian cancer. Expert Rev Proteomics. 2007 February; 4(1):121-31;Carney, Circulating oncoproteins HER2/neu, EGFR and CAIX (MN) as novelcancer biomarkers. Expert Rev Mol. Diagn. 2007 May; 7(3):309-19; Gagnon,Discovery and application of protein biomarkers for ovarian cancer, CurrOpin Obstet. Gynecol. 2008 February; 20(1):9-13; Pasterkamp et al,Immune regulatory cells: circulating biomarker factories incardiovascular disease. Clin Sci (Loud). 2008 August; 115(4):129-31;Fabbri, miRNAs as molecular biomarkers of cancer, Exp Rev Mol Diag, May2010, Vol. 10, No. 4, Pages 435-444; PCT Patent PublicationWO/2007/088537; U.S. Pat. Nos. 7,745,150 and 7,655,479; U.S. PatentPublications 20110008808, 20100330683, 20100248290, 20100222230,20100203566, 20100173788, 20090291932, 20090239246, 20090226937,20090111121, 20090004687, 20080261258, 20080213907, 20060003465,20050124071, and 20040096915, each of which publication is incorporatedherein by reference in its entirety. In an embodiment, molecularprofiling of the invention comprises analysis of circulating biomarkers.

Gene Expression Profiling

The methods and systems of the invention comprise expression profiling,which includes assessing differential expression of one or more targetgenes disclosed herein. Differential expression can includeoverexpression and/or underexpression of a biological product, e.g., agene, mRNA or protein, compared to a control (or a reference). Thecontrol can include similar cells to the sample but without the disease(e.g., expression profiles obtained from samples from healthyindividuals). A control can be a previously determined level that isindicative of a drug target efficacy associated with the particulardisease and the particular drug target. The control can be derived fromthe same patient, e.g., a normal adjacent portion of the same organ asthe diseased cells, the control can be derived from healthy tissues fromother patients, or previously determined thresholds that are indicativeof a disease responding or not-responding to a particular drug target.The control can also be a control found in the same sample, e.g. ahousekeeping gene or a product thereof (e.g., mRNA or protein). Forexample, a control nucleic acid can be one which is known not to differdepending on the cancerous or non-cancerous state of the cell. Theexpression level of a control nucleic acid can be used to normalizesignal levels in the test and reference populations. Illustrativecontrol genes include, but are not limited to, e.g., β-actin,glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein P1.Multiple controls or types of controls can be used. The source ofdifferential expression can vary. For example, a gene copy number may beincreased in a cell, thereby resulting in increased expression of thegene. Alternately, transcription of the gene may be modified, e.g., bychromatin remodeling, differential methylation, differential expressionor activity of transcription factors, etc. Translation may also bemodified, e.g., by differential expression of factors that degrade mRNA,translate mRNA, or silence translation, e.g., microRNAs or siRNAs. Insome embodiments, differential expression comprises differentialactivity. For example, a protein may carry a mutation that increases theactivity of the protein, such as constitutive activation, therebycontributing to a diseased state. Molecular profiling that revealschanges in activity can be used to guide treatment selection.

Methods of gene expression profiling include methods based onhybridization analysis of polynucleotides, and methods based onsequencing of polynucleotides. Commonly used methods known in the artfor the quantification of mRNA expression in a sample include northernblotting and in situ hybridization (Parker & Barnes (1999) Methods inMolecular Biology 106:247-283); RNAse protection assays (Hod (1992)Biotechniques 13:852-854); and reverse transcription polymerase chainreaction (RT-PCR) (Weis et al. (1992) Trends in Genetics 8:263-264).Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. Representative methods forsequencing-based gene expression analysis include Serial Analysis ofGene Expression (SAGE), gene expression analysis by massively parallelsignature sequencing (MPSS) and/or next generation sequencing.

Real time PCR (RT-PCR)

RT-PCR can be used to determine RNA levels, e.g., mRNA or miRNA levels,of the biomarkers of the invention. RT-PCR can be used to compare suchRNA levels of the biomarkers of the invention in different samplepopulations, in normal and tumor tissues, with or without drugtreatment, to characterize patterns of gene expression, to discriminatebetween closely related RNAs, and to analyze RNA structure.

The first step is the isolation of RNA, e.g., mRNA, from a sample. Thestarting material can be total RNA isolated from human tumors or tumorcell lines, and corresponding normal tissues or cell lines,respectively. Thus RNA can be isolated from a sample, e.g., tumor cellsor tumor cell lines, and compared with pooled DNA from healthy donors.If the source of mRNA is a primary tumor, mRNA can be extracted, forexample, from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples.

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al. (1997) Current Protocols of Molecular Biology, John Wiley andSons. Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, andDe Andres et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer's instructions (QIAGEN Inc., Valencia, Calif.). Forexample, total RNA from cells in culture can be isolated using QiagenRNeasy mini-columns. Numerous RNA isolation kits are commerciallyavailable and can be used in the methods of the invention.

In the alternative, the first step is the isolation of miRNA from atarget sample. The starting material is typically total RNA isolatedfrom human tumors or tumor cell lines, and corresponding normal tissuesor cell lines, respectively. Thus RNA can be isolated from a variety ofprimary tumors or tumor cell lines, with pooled DNA from healthy donors.If the source of miRNA is a primary tumor, miRNA can be extracted, forexample, from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples.

General methods for miRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al. (1997) Current Protocols of Molecular Biology, John Wiley andSons. Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, andDe Andres et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Numerous RNAisolation kits are commercially available and can be used in the methodsof the invention.

Whether the RNA comprises mRNA, miRNA or other types of RNA, geneexpression profiling by RT-PCR can include reverse transcription of theRNA template into cDNA, followed by amplification in a PCR reaction.Commonly used reverse transcriptases include, but are not limited to,avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloneymurine leukemia virus reverse transcriptase (MMLV-RT). The reversetranscription step is typically primed using specific primers, randomhexamers, or oligo-dT primers, depending on the circumstances and thegoal of expression profiling. For example, extracted RNA can bereverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif.,USA), following the manufacturer's instructions. The derived cDNA canthen be used as a template in the subsequent PCR reaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. TaqMan PCR typically uses the 5′-nuclease activity of Taq orTth polymerase to hydrolyze a hybridization probe bound to its targetamplicon, but any enzyme with equivalent 5′ nuclease activity can beused. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan™ RT-PCR can be performed using commercially available equipment,such as, for example, ABI PRISM 7700™ Sequence Detection System™(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightCycler (Roche Molecular Biochemicals, Mannheim, Germany). In onespecific embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRISM 7700 Sequence DetectionSystem. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optic cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

TaqMan data are initially expressed as Ct, or the threshold cycle. Asdiscussed above, fluorescence values are recorded during every cycle andrepresent the amount of product amplified to that point in theamplification reaction. The point when the fluorescent signal is firstrecorded as statistically significant is the threshold cycle (Ct).

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.

Real time quantitative PCR (also quantitative real time polymerase chainreaction, QRT-PCR or Q-PCR) is a more recent variation of the RT-PCRtechnique. Q-PCR can measure PCR product accumulation through adual-labeled fluorigenic probe (i.e., TaqMan probe). Real time PCR iscompatible both with quantitative competitive PCR, where internalcompetitor for each target sequence is used for normalization, and withquantitative comparative PCR using a normalization gene contained withinthe sample, or a housekeeping gene for RT-PCR. See, e.g. Held et al.(1996) Genome Research 6:986-994.

Protein-based detection techniques are also useful for molecularprofiling, especially when the nucleotide variant causes amino acidsubstitutions or deletions or insertions or frame shift that affect theprotein primary, secondary or tertiary structure. To detect the aminoacid variations, protein sequencing techniques may be used. For example,a protein or fragment thereof corresponding to a gene can be synthesizedby recombinant expression using a DNA fragment isolated from anindividual to be tested. Preferably, a cDNA fragment of no more than 100to 150 base pairs encompassing the polymorphic locus to be determined isused. The amino acid sequence of the peptide can then be determined byconventional protein sequencing methods. Alternatively, theHPLC-microscopy tandem mass spectrometry technique can be used fordetermining the amino acid sequence variations. In this technique,proteolytic digestion is performed on a protein, and the resultingpeptide mixture is separated by reversed-phase chromatographicseparation. Tandem mass spectrometry is then performed and the datacollected is analyzed. See Gatlin et al., Anal. Chem., 72:757-763(2000).

Microarray

The biomarkers of the invention can also be identified, confirmed,and/or measured using the microarray technique. Thus, the expressionprofile biomarkers can be measured in cancer samples using microarraytechnology. In this method, polynucleotide sequences of interest areplated, or arrayed, on a microchip substrate. The arrayed sequences arethen hybridized with specific DNA probes from cells or tissues ofinterest. The source of mRNA can be total RNA isolated from a sample,e.g., human tumors or tumor cell lines and corresponding normal tissuesor cell lines. Thus RNA can be isolated from a variety of primary tumorsor tumor cell lines. If the source of mRNA is a primary tumor, mRNA canbe extracted, for example, from frozen or archived paraffin-embedded andfixed (e.g. formalin-fixed) tissue samples, which are routinely preparedand preserved in everyday clinical practice.

The expression profile of biomarkers can be measured in either fresh orparaffin-embedded tumor tissue, or body fluids using microarraytechnology. In this method, polynucleotide sequences of interest areplated, or arrayed, on a microchip substrate. The arrayed sequences arethen hybridized with specific DNA probes from cells or tissues ofinterest. As with the RT-PCR method, the source of miRNA typically istotal RNA isolated from human tumors or tumor cell lines, including bodyfluids, such as serum, urine, tears, and exosomes and correspondingnormal tissues or cell lines. Thus RNA can be isolated from a variety ofsources. If the source of miRNA is a primary tumor, miRNA can beextracted, for example, from frozen tissue samples, which are routinelyprepared and preserved in everyday clinical practice.

Also known as biochip, DNA chip, or gene array, cDNA microarraytechnology allows for identification of gene expression levels in abiologic sample. cDNAs or oligonucleotides, each representing a givengene, are immobilized on a substrate, e.g., a small chip, bead or nylonmembrane, tagged, and serve as probes that will indicate whether theyare expressed in biologic samples of interest. The simultaneousexpression of thousands of genes can be monitored simultaneously.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate in a dense array. Inone aspect, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000,1,500, 2,000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000,20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or at least 50,000nucleotide sequences are applied to the substrate. Each sequence cancorrespond to a different gene, or multiple sequences can be arrayed pergene. The microarrayed genes, immobilized on the microchip, are suitablefor hybridization under stringent conditions. Fluorescently labeled cDNAprobes may be generated through incorporation of fluorescent nucleotidesby reverse transcription of RNA extracted from tissues of interest.Labeled cDNA probes applied to the chip hybridize with specificity toeach spot of DNA on the array. After stringent washing to removenon-specifically bound probes, the chip is scanned by confocal lasermicroscopy or by another detection method, such as a CCD camera.Quantitation of hybridization of each arrayed element allows forassessment of corresponding mRNA abundance. With dual colorfluorescence, separately labeled cDNA probes generated from two sourcesof RNA are hybridized pairwise to the array. The relative abundance ofthe transcripts from the two sources corresponding to each specifiedgene is thus determined simultaneously. The miniaturized scale of thehybridization affords a convenient and rapid evaluation of theexpression pattern for large numbers of genes. Such methods have beenshown to have the sensitivity required to detect rare transcripts, whichare expressed at a few copies per cell, and to reproducibly detect atleast approximately two-fold differences in the expression levels(Schena et al. (1996) Proc. Natl. Acad. Sci. USA 93(2):106-149).Microarray analysis can be performed by commercially available equipmentfollowing manufacturer's protocols, including without limitation theAffymetrix GeneChip technology (Affymetrix, Santa Clara, Calif.),Agilent (Agilent Technologies, Inc., Santa Clara, Calif.), or Illumina(Illumina, Inc., San Diego, Calif.) microarray technology.

The development of microarray methods for large-scale analysis of geneexpression makes it possible to search systematically for molecularmarkers of cancer classification and outcome prediction in a variety oftumor types.

In some embodiments, the Agilent Whole Human Genome Microarray Kit(Agilent Technologies, Inc., Santa Clara, Calif.). The system cananalyze more than 41,000 unique human genes and transcripts represented,all with public domain annotations. The system is used according to themanufacturer's instructions.

In some embodiments, the Illumina Whole Genome DASL assay (IlluminaInc., San Diego, Calif.) is used. The system offers a method tosimultaneously profile over 24,000 transcripts from minimal RNA input,from both fresh frozen (FF) and formalin-fixed paraffin embedded (FFPE)tissue sources, in a high throughput fashion.

Microarray expression analysis comprises identifying whether a gene orgene product is up-regulated or down-regulated relative to a reference.The identification can be performed using a statistical test todetermine statistical significance of any differential expressionobserved. In some embodiments, statistical significance is determinedusing a parametric statistical test. The parametric statistical test cancomprise, for example, a fractional factorial design, analysis ofvariance (ANOVA), a t-test, least squares, a Pearson correlation, simplelinear regression, nonlinear regression, multiple linear regression, ormultiple nonlinear regression. Alternatively, the parametric statisticaltest can comprise a one-way analysis of variance, two-way analysis ofvariance, or repeated measures analysis of variance. In otherembodiments, statistical significance is determined using anonparametric statistical test. Examples include, but are not limitedto, a Wilcoxon signed-rank test, a Mann-Whitney test, a Kruskal-Wallistest, a Friedman test, a Spearman ranked order correlation coefficient,a Kendall Tau analysis, and a nonparametric regression test. In someembodiments, statistical significance is determined at a p-value of lessthan about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. Although themicroarray systems used in the methods of the invention may assaythousands of transcripts, data analysis need only be performed on thetranscripts of interest, thereby reducing the problem of multiplecomparisons inherent in performing multiple statistical tests. Thep-values can also be corrected for multiple comparisons, e.g., using aBonferroni correction, a modification thereof, or other technique knownto those in the art, e.g., the Hochberg correction, Holm-Bonferronicorrection, Sidak correction, or Dunnett's correction. The degree ofdifferential expression can also be taken into account. For example, agene can be considered as differentially expressed when the fold-changein expression compared to control level is at least 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-folddifferent in the sample versus the control. The differential expressiontakes into account both overexpression and underexpression. A gene orgene product can be considered up or down-regulated if the differentialexpression meets a statistical threshold, a fold-change threshold, orboth. For example, the criteria for identifying differential expressioncan comprise both a p-value of 0.001 and fold change of at least1.5-fold (up or down). One of skill will understand that suchstatistical and threshold measures can be adapted to determinedifferential expression by any molecular profiling technique disclosedherein.

Various methods of the invention make use of many types of microarraysthat detect the presence and potentially the amount of biologicalentities in a sample. Arrays typically contain addressable moieties thatcan detect the presence of the entity in the sample, e.g., via a bindingevent. Microarrays include without limitation DNA microarrays, such ascDNA microarrays, oligonucleotide microarrays and SNP microarrays,microRNA arrays, protein microarrays, antibody microarrays, tissuemicroarrays, cellular microarrays (also called transfectionmicroarrays), chemical compound microarrays, and carbohydrate arrays(glycoarrays). DNA arrays typically comprise addressable nucleotidesequences that can bind to sequences present in a sample. MicroRNAarrays, e.g., the MMChips array from the University of Louisville orcommercial systems from Agilent, can be used to detect microRNAs.Protein microarrays can be used to identify protein-proteininteractions, including without limitation identifying substrates ofprotein kinases, transcription factor protein-activation, or to identifythe targets of biologically active small molecules. Protein arrays maycomprise an array of different protein molecules, commonly antibodies,or nucleotide sequences that bind to proteins of interest. Antibodymicroarrays comprise antibodies spotted onto the protein chip that areused as capture molecules to detect proteins or other biologicalmaterials from a sample, e.g., from cell or tissue lysate solutions. Forexample, antibody arrays can be used to detect biomarkers from bodilyfluids, e.g., serum or urine, for diagnostic applications. Tissuemicroarrays comprise separate tissue cores assembled in array fashion toallow multiplex histological analysis. Cellular microarrays, also calledtransfection microarrays, comprise various capture agents, such asantibodies, proteins, or lipids, which can interact with cells tofacilitate their capture on addressable locations. Chemical compoundmicroarrays comprise arrays of chemical compounds and can be used todetect protein or other biological materials that bind the compounds.Carbohydrate arrays (glycoarrays) comprise arrays of carbohydrates andcan detect, e.g., protein that bind sugar moieties. One of skill willappreciate that similar technologies or improvements can be usedaccording to the methods of the invention.

Certain embodiments of the current methods comprise a multi-wellreaction vessel, including without limitation, a multi-well plate or amulti-chambered microfluidic device, in which a multiplicity ofamplification reactions and, in some embodiments, detection areperformed, typically in parallel. In certain embodiments, one or moremultiplex reactions for generating amplicons are performed in the samereaction vessel, including without limitation, a multi-well plate, suchas a 96-well, a 384-well, a 1536-well plate, and so forth; or amicrofluidic device, for example but not limited to, a TaqMan™ LowDensity Array (Applied Biosystems, Foster City, Calif.). In someembodiments, a massively parallel amplifying step comprises a multi-wellreaction vessel, including a plate comprising multiple reaction wells,for example but not limited to, a 24-well plate, a 96-well plate, a384-well plate, or a 1536-well plate; or a multi-chamber microfluidicsdevice, for example but not limited to a low density array wherein eachchamber or well comprises an appropriate primer(s), primer set(s),and/or reporter probe(s), as appropriate. Typically such amplificationsteps occur in a series of parallel single-plex, two-plex, three-plex,four-plex, five-plex, or six-plex reactions, although higher levels ofparallel multiplexing are also within the intended scope of the currentteachings. These methods can comprise PCR methodology, such as RT-PCR,in each of the wells or chambers to amplify and/or detect nucleic acidmolecules of interest.

Low density arrays can include arrays that detect 10s or 100s ofmolecules as opposed to 1000s of molecules. These arrays can be moresensitive than high density arrays. In embodiments, a low density arraysuch as a TaqMan™ Low Density Array is used to detect one or more geneor gene product in Table 2. For example, the low density array can beused to detect at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90 or 100 genes or gene products in Table 2.

In some embodiments, the disclosed methods comprise a microfluidicsdevice, “lab on a chip,” or micrototal analytical system (pTAS). In someembodiments, sample preparation is performed using a microfluidicsdevice. In some embodiments, an amplification reaction is performedusing a microfluidics device. In some embodiments, a sequencing or PCRreaction is performed using a microfluidic device. In some embodiments,the nucleotide sequence of at least a part of an amplified product isobtained using a microfluidics device. In some embodiments, detectingcomprises a microfluidic device, including without limitation, a lowdensity array, such as a TaqMan™ Low Density Array. Descriptions ofexemplary microfluidic devices can be found in, among other places,Published PCT Application Nos. WO/0185341 and WO 04/011666; Kartalov andQuake, Nucl. Acids Res. 32:2873-79, 2004; and Fiorini and Chiu, BioTechniques 38:429-46, 2005.

Any appropriate microfluidic device can be used in the methods of theinvention. Examples of microfluidic devices that may be used, or adaptedfor use with molecular profiling, include but are not limited to thosedescribed in U.S. Pat. Nos. 7,591,936, 7,581,429, 7,579,136, 7,575,722,7,568,399, 7,552,741, 7,544,506, 7,541,578, 7,518,726, 7,488,596,7,485,214, 7,467,928, 7,452,713, 7,452,509, 7,449,096, 7,431,887,7,422,725, 7,422,669, 7,419,822, 7,419,639, 7,413,709, 7,411,184,7,402,229, 7,390,463, 7,381,471, 7,357,864, 7,351,592, 7,351,380,7,338,637, 7,329,391, 7,323,140, 7,261,824, 7,258,837, 7,253,003,7,238,324, 7,238,255, 7,233,865, 7,229,538, 7,201,881, 7,195,986,7,189,581, 7,189,580, 7,189,368, 7,141,978, 7,138,062, 7,135,147,7,125,711, 7,118,910, 7,118,661, 7,640,947, 7,666,361, 7,704,735; U.S.Patent Application Publication 20060035243; and International PatentPublication WO 2010/072410; each of which patents or applications areincorporated herein by reference in their entirety. Another example foruse with methods disclosed herein is described in Chen et al.,“Microfluidic isolation and transcriptome analysis of serum vesicles,”Lab on a Chip, Dec. 8, 2009 DOI: 10.1039/b916199f.

Gene Expression Analysis by Massively Parallel Signature Sequencing(MPSS)

This method, described by Brenner et al. (2000) Nature Biotechnology18:630-634, is a sequencing approach that combines non-gel-basedsignature sequencing with in vitro cloning of millions of templates onseparate microbeads. First, a microbead library of DNA templates isconstructed by in vitro cloning. This is followed by the assembly of aplanar array of the template-containing microbeads in a flow cell at ahigh density. The free ends of the cloned templates on each microbeadare analyzed simultaneously, using a fluorescence-based signaturesequencing method that does not require DNA fragment separation. Thismethod has been shown to simultaneously and accurately provide, in asingle operation, hundreds of thousands of gene signature sequences froma cDNA library.

MPSS data has many uses. The expression levels of nearly all transcriptscan be quantitatively determined; the abundance of signatures isrepresentative of the expression level of the gene in the analyzedtissue. Quantitative methods for the analysis of tag frequencies anddetection of differences among libraries have been published andincorporated into public databases for SAGE™ data and are applicable toMPSS data. The availability of complete genome sequences permits thedirect comparison of signatures to genomic sequences and further extendsthe utility of MPSS data. Because the targets for MPSS analysis are notpre-selected (like on a microarray), MPSS data can characterize the fullcomplexity of transcriptomes. This is analogous to sequencing millionsof ESTs at once, and genomic sequence data can be used so that thesource of the MPSS signature can be readily identified by computationalmeans.

Serial Analysis of Gene Expression (SAGE)

Serial analysis of gene expression (SAGE) is a method that allows thesimultaneous and quantitative analysis of a large number of genetranscripts, without the need of providing an individual hybridizationprobe for each transcript. First, a short sequence tag (e.g., about10-14 bp) is generated that contains sufficient information to uniquelyidentify a transcript, provided that the tag is obtained from a uniqueposition within each transcript. Then, many transcripts are linkedtogether to form long serial molecules, that can be sequenced, revealingthe identity of the multiple tags simultaneously. The expression patternof any population of transcripts can be quantitatively evaluated bydetermining the abundance of individual tags, and identifying the genecorresponding to each tag. See, e.g. Velculescu et al. (1995) Science270:484-487; and Velculescu et al. (1997) Cell 88:243-51.

DNA Copy Number Profiling

Any method capable of determining a DNA copy number profile of aparticular sample can be used for molecular profiling according to theinvention as long as the resolution is sufficient to identify thebiomarkers of the invention. The skilled artisan is aware of and capableof using a number of different platforms for assessing whole genome copynumber changes at a resolution sufficient to identify the copy number ofthe one or more biomarkers of the invention. Some of the platforms andtechniques are described in the embodiments below.

In some embodiments, the copy number profile analysis involvesamplification of whole genome DNA by a whole genome amplificationmethod. The whole genome amplification method can use a stranddisplacing polymerase and random primers.

In some aspects of these embodiments, the copy number profile analysisinvolves hybridization of whole genome amplified DNA with a high densityarray. In a more specific aspect, the high density array has 5,000 ormore different probes. In another specific aspect, the high densityarray has 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000,400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 ormore different probes. In another specific aspect, each of the differentprobes on the array is an oligonucleotide having from about 15 to 200bases in length. In another specific aspect, each of the differentprobes on the array is an oligonucleotide having from about 15 to 200,15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length.

In some embodiments, a microarray is employed to aid in determining thecopy number profile for a sample, e.g., cells from a tumor. Microarraystypically comprise a plurality of oligomers (e.g., DNA or RNApolynucleotides or oligonucleotides, or other polymers), synthesized ordeposited on a substrate (e.g., glass support) in an array pattern. Thesupport-bound oligomers are “probes”, which function to hybridize orbind with a sample material (e.g., nucleic acids prepared or obtainedfrom the tumor samples), in hybridization experiments. The reversesituation can also be applied: the sample can be bound to the microarraysubstrate and the oligomer probes are in solution for the hybridization.In use, the array surface is contacted with one or more targets underconditions that promote specific, high-affinity binding of the target toone or more of the probes. In some configurations, the sample nucleicacid is labeled with a detectable label, such as a fluorescent tag, sothat the hybridized sample and probes are detectable with scanningequipment. DNA array technology offers the potential of using amultitude (e.g., hundreds of thousands) of different oligonucleotides toanalyze DNA copy number profiles. In some embodiments, the substratesused for arrays are surface-derivatized glass or silica, or polymermembrane surfaces (see e.g., in Z. Guo, et al., Nucleic Acids Res, 22,5456-65 (1994); U. Maskos, E. M. Southern, Nucleic Acids Res, 20,1679-84 (1992), and E. M. Southern, et al., Nucleic Acids Res, 22,1368-73 (1994), each incorporated by reference herein). Modification ofsurfaces of array substrates can be accomplished by many techniques. Forexample, siliceous or metal oxide surfaces can be derivatized withbifunctional silanes, i.e., silanes having a first functional groupenabling covalent binding to the surface (e.g., Si-halogen or Si-alkoxygroup, as in —SiCl₃ or —Si(OCH₃)₃, respectively) and a second functionalgroup that can impart the desired chemical and/or physical modificationsto the surface to covalently or non-covalently attach ligands and/or thepolymers or monomers for the biological probe array. Silylatedderivatizations and other surface derivatizations that are known in theart (see for example U.S. Pat. No. 5,624,711 to Sundberg, U.S. Pat. No.5,266,222 to Willis, and U.S. Pat. No. 5,137,765 to Farnsworth, eachincorporated by reference herein). Other processes for preparing arraysare described in U.S. Pat. No. 6,649,348, to Bass et. al., assigned toAgilent Corp., which disclose DNA arrays created by in situ synthesismethods.

Polymer array synthesis is also described extensively in the literatureincluding in the following: WO 00/58516, U.S. Pat. Nos. 5,143,854,5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186,5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639,5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716,5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740,5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193,6,090,555, 6,136,269, 6,269,846 and 6,428,752, 5,412,087, 6,147,205,6,262,216, 6,310,189, 5,889,165, and 5,959,098 in PCT Applications Nos.PCT/US99/00730 (International Publication No. WO 99/36760) andPCT/US01/04285 (International Publication No. WO 01/58593), which areall incorporated herein by reference in their entirety for all purposes.

Nucleic acid arrays that are useful in the present invention include,but are not limited to, those that are commercially available fromAffymetrix (Santa Clara, Calif.) under the brand name GeneChip™. Examplearrays are shown on the website at affymetrix.com. Another microarraysupplier is Illumina, Inc., of San Diego, Calif. with example arraysshown on their website at illumina.com.

In some embodiments, the inventive methods provide for samplepreparation. Depending on the microarray and experiment to be performed,sample nucleic acid can be prepared in a number of ways by methods knownto the skilled artisan. In some aspects of the invention, prior to orconcurrent with genotyping (analysis of copy number profiles), thesample may be amplified any number of mechanisms. The most commonamplification procedure used involves PCR. See, for example, PCRTechnology: Principles and Applications for DNA Amplification (Ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (Eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991);Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds.McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202,4,683,195, 4,800,159 4,965,188, and 5,333,675, and each of which isincorporated herein by reference in their entireties for all purposes.In some embodiments, the sample may be amplified on the array (e.g.,U.S. Pat. No. 6,300,070 which is incorporated herein by reference)

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent ApplicationPublication 20030096235), 09/910,292 (U.S. Patent ApplicationPublication 20030082543), and 10/013,598.

Methods for conducting polynucleotide hybridization assays are welldeveloped in the art. Hybridization assay procedures and conditions usedin the methods of the invention will vary depending on the applicationand are selected in accordance with the general binding methods knownincluding those referred to in: Maniatis et al. Molecular Cloning: ALaboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y., 1989); Bergerand Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular CloningTechniques (Academic Press, Inc., San Diego, Calif., 1987); Young andDavism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying outrepeated and controlled hybridization reactions have been described inU.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623each of which are incorporated herein by reference.

The methods of the invention may also involve signal detection ofhybridization between ligands in after (and/or during) hybridization.See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCTApplication PCT/US99/06097 (published as WO99/47964), each of which alsois hereby incorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,60/493,495 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

Immuno-Based Assays

Protein-based detection molecular profiling techniques includeimmunoaffinity assays based on antibodies selectively immunoreactivewith mutant gene encoded protein according to the present invention.These techniques include without limitation immunoprecipitation, Westernblot analysis, molecular binding assays, enzyme-linked immunosorbentassay (ELISA), enzyme-linked immunofiltration assay (ELIFA),fluorescence activated cell sorting (FACS) and the like. For example, anoptional method of detecting the expression of a biomarker in a samplecomprises contacting the sample with an antibody against the biomarker,or an immunoreactive fragment of the antibody thereof, or a recombinantprotein containing an antigen binding region of an antibody against thebiomarker; and then detecting the binding of the biomarker in thesample. Methods for producing such antibodies are known in the art.Antibodies can be used to immunoprecipitate specific proteins fromsolution samples or to immunoblot proteins separated by, e.g.,polyacrylamide gels. Immunocytochemical methods can also be used indetecting specific protein polymorphisms in tissues or cells. Otherwell-known antibody-based techniques can also be used including, e.g.,ELISA, radioimmunoassay (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal or polyclonal antibodies. See, e.g., U.S. Pat. Nos. 4,376,110and 4,486,530, both of which are incorporated herein by reference.

In alternative methods, the sample may be contacted with an antibodyspecific for a biomarker under conditions sufficient for anantibody-biomarker complex to form, and then detecting the complex. Thepresence of the biomarker may be detected in a number of ways, such asby Western blotting and ELISA procedures for assaying a wide variety oftissues and samples, including plasma or serum. A wide range ofimmunoassay techniques using such an assay format are available, see,e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labelled antibodyto a target biomarker.

A number of variations of the sandwich assay technique exist, and allare intended to be encompassed by the present invention. Briefly, in atypical forward assay, an unlabelled antibody is immobilized on a solidsubstrate, and the sample to be tested brought into contact with thebound molecule. After a suitable period of incubation, for a period oftime sufficient to allow formation of an antibody-antigen complex, asecond antibody specific to the antigen, labelled with a reportermolecule capable of producing a detectable signal is then added andincubated, allowing time sufficient for the formation of another complexof antibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof biomarker.

Variations on the forward assay include a simultaneous assay, in whichboth sample and labelled antibody are added simultaneously to the boundantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent. In a typicalforward sandwich assay, a first antibody having specificity for thebiomarker is either covalently or passively bound to a solid surface.The solid surface is typically glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene. The solid supports may be in theform of tubes, beads, discs of microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking covalentlybinding or physically adsorbing, the polymer-antibody complex is washedin preparation for the test sample. An aliquot of the sample to betested is then added to the solid phase complex and incubated for aperiod of time sufficient (e.g. 2-40 minutes or overnight if moreconvenient) and under suitable conditions (e.g. from room temperature to40° C. such as between 25° C. and 32° C. inclusive) to allow binding ofany subunit present in the antibody. Following the incubation period,the antibody subunit solid phase is washed and dried and incubated witha second antibody specific for a portion of the biomarker. The secondantibody is linked to a reporter molecule which is used to indicate thebinding of the second antibody to the molecular marker.

An alternative method involves immobilizing the target biomarkers in thesample and then exposing the immobilized target to specific antibodywhich may or may not be labelled with a reporter molecule. Depending onthe amount of target and the strength of the reporter molecule signal, abound target may be detectable by direct labelling with the antibody.Alternatively, a second labelled antibody, specific to the firstantibody is exposed to the target-first antibody complex to form atarget-first antibody-second antibody tertiary complex. The complex isdetected by the signal emitted by the reporter molecule. By “reportermolecule”, as used in the present specification, is meant a moleculewhich, by its chemical nature, provides an analytically identifiablesignal which allows the detection of antigen-bound antibody. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, β-galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody-molecular marker complex,allowed to bind, and then the excess reagent is washed away. A solutioncontaining the appropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of biomarker which was present in the sample.Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-molecular marker complex. After washingoff the unbound reagent, the remaining tertiary complex is then exposedto the light of the appropriate wavelength, the fluorescence observedindicates the presence of the molecular marker of interest.Immunofluorescence and EIA techniques are both very well established inthe art. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules, may also be employed.

Immunohistochemistry (IHC)

IHC is a process of localizing antigens (e.g., proteins) in cells of atissue binding antibodies specifically to antigens in the tissues. Theantigen-binding antibody can be conjugated or fused to a tag that allowsits detection, e.g., via visualization. In some embodiments, the tag isan enzyme that can catalyze a color-producing reaction, such as alkalinephosphatase or horseradish peroxidase. The enzyme can be fused to theantibody or non-covalently bound, e.g., using a biotin-avadin system.Alternatively, the antibody can be tagged with a fluorophore, such asfluorescein, rhodamine, DyLight Fluor or Alexa Fluor. Theantigen-binding antibody can be directly tagged or it can itself berecognized by a detection antibody that carries the tag. Using IHC, oneor more proteins may be detected. The expression of a gene product canbe related to its staining intensity compared to control levels. In someembodiments, the gene product is considered differentially expressed ifits staining varies at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold in the sampleversus the control.

IHC comprises the application of antigen-antibody interactions tohistochemical techniques. In an illustrative example, a tissue sectionis mounted on a slide and is incubated with antibodies (polyclonal ormonoclonal) specific to the antigen (primary reaction). Theantigen-antibody signal is then amplified using a second antibodyconjugated to a complex of peroxidase antiperoxidase (PAP),avidin-biotin-peroxidase (ABC) or avidin-biotin alkaline phosphatase. Inthe presence of substrate and chromogen, the enzyme forms a coloreddeposit at the sites of antibody-antigen binding. Immunofluorescence isan alternate approach to visualize antigens. In this technique, theprimary antigen-antibody signal is amplified using a second antibodyconjugated to a fluorochrome. On UV light absorption, the fluorochromeemits its own light at a longer wavelength (fluorescence), thus allowinglocalization of antibody-antigen complexes.

Epigenetic Status

Molecular profiling methods according to the invention also comprisemeasuring epigenetic change, i.e., modification in a gene caused by anepigenetic mechanism, such as a change in methylation status or histoneacetylation. Frequently, the epigenetic change will result in analteration in the levels of expression of the gene which may be detected(at the RNA or protein level as appropriate) as an indication of theepigenetic change. Often the epigenetic change results in silencing ordown regulation of the gene, referred to as “epigenetic silencing.” Themost frequently investigated epigenetic change in the methods of theinvention involves determining the DNA methylation status of a gene,where an increased level of methylation is typically associated with therelevant cancer (since it may cause down regulation of gene expression).Aberrant methylation, which may be referred to as hypermethylation, ofthe gene or genes can be detected. Typically, the methylation status isdetermined in suitable CpG islands which are often found in the promoterregion of the gene(s). The term “methylation,” “methylation state” or“methylation status” may refers to the presence or absence of5-methylcytosine at one or a plurality of CpG dinucleotides within a DNAsequence. CpG dinucleotides are typically concentrated in the promoterregions and exons of human genes.

Diminished gene expression can be assessed in terms of DNA methylationstatus or in terms of expression levels as determined by the methylationstatus of the gene. One method to detect epigenetic silencing is todetermine that a gene which is expressed in normal cells is lessexpressed or not expressed in tumor cells. Accordingly, the inventionprovides for a method of molecular profiling comprising detectingepigenetic silencing.

Various assay procedures to directly detect methylation are known in theart, and can be used in conjunction with the present invention. Theseassays rely onto two distinct approaches: bisulphite conversion basedapproaches and non-bisulphite based approaches. Non-bisulphite basedmethods for analysis of DNA methylation rely on the inability ofmethylation-sensitive enzymes to cleave methylation cytosines in theirrestriction. The bisulphite conversion relies on treatment of DNAsamples with sodium bisulphite which converts unmethylated cytosine touracil, while methylated cytosines are maintained (Furuichi Y, Wataya Y,Hayatsu H, Ukita T. Biochem Biophys Res Commun. 1970 Dec. 9;41(5):1185-91). This conversion results in a change in the sequence ofthe original DNA. Methods to detect such changes include MS AP-PCR(Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction), atechnology that allows for a global scan of the genome using CG-richprimers to focus on the regions most likely to contain CpGdinucleotides, and described by Gonzalgo et al., Cancer Research57:594-599, 1997; MethyLight™, which refers to the art-recognizedfluorescence-based real-time PCR technique described by Eads et al.,Cancer Res. 59:2302-2306, 1999; the HeavyMethyl™assay, in the embodimentthereof implemented herein, is an assay, wherein methylation specificblocking probes (also referred to herein as blockers) covering CpGpositions between, or covered by the amplification primers enablemethylation-specific selective amplification of a nucleic acid sample;HeavyMethyl™MethyLight™ is a variation of the MethyLight™ assay whereinthe MethyLight™ assay is combined with methylation specific blockingprobes covering CpG positions between the amplification primers;Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) isan assay described by Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531,1997; MSP (Methylation-specific PCR) is a methylation assay described byHerman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, and by U.S.Pat. No. 5,786,146; COBRA (Combined Bisulfite Restriction Analysis) is amethylation assay described by Xiong & Laird, Nucleic Acids Res.25:2532-2534, 1997; MCA (Methylated CpG Island Amplification) is amethylation assay described by Toyota et al., Cancer Res. 59:2307-12,1999, and in WO 00/26401A1.

Other techniques for DNA methylation analysis include sequencing,methylation-specific PCR (MS-PCR), melting curve methylation-specificPCR (McMS-PCR), MLPA with or without bisulfite treatment, QAMA,MSRE-PCR, MethyLight, ConLight-MSP, bisulfite conversion-specificmethylation-specific PCR (BS-MSP), COBRA (which relies upon use ofrestriction enzymes to reveal methylation dependent sequence differencesin PCR products of sodium bisulfite-treated DNA), methylation-sensitivesingle-nucleotide primer extension conformation (MS-SNuPE),methylation-sensitive single-strand conformation analysis (MS-SSCA),Melting curve combined bisulfite restriction analysis (McCOBRA),PyroMethA, HeavyMethyl, MALDI-TOF, MassARRAY, Quantitative analysis ofmethylated alleles (QAMA), enzymatic regional methylation assay (ERMA),QBSUPT, MethylQuant, Quantitative PCR sequencing andoligonucleotide-based microarray systems, Pyrosequencing, Meth-DOP-PCR.A review of some useful techniques is provided in Nucleic acidsresearch, 1998, Vol. 26, No. 10, 2255-2264; Nature Reviews, 2003, Vol.3, 253-266; Oral Oncology, 2006, Vol. 42, 5-13, which references areincorporated herein in their entirety. Any of these techniques may beused in accordance with the present invention, as appropriate. Othertechniques are described in U.S. Patent Publications 20100144836; and20100184027, which applications are incorporated herein by reference intheir entirety.

Through the activity of various acetylases and deacetylylases the DNAbinding function of histone proteins is tightly regulated. Furthermore,histone acetylation and histone deactelyation have been linked withmalignant progression. See Nature, 429: 457-63, 2004. Methods to analyzehistone acetylation are described in U.S. Patent Publications20100144543 and 20100151468, which applications are incorporated hereinby reference in their entirety.

Sequence Analysis

Molecular profiling according to the present invention comprises methodsfor genotyping one or more biomarkers by determining whether anindividual has one or more nucleotide variants (or amino acid variants)in one or more of the genes or gene products. Genotyping one or moregenes according to the methods of the invention in some embodiments, canprovide more evidence for selecting a treatment.

The biomarkers of the invention can be analyzed by any method useful fordetermining alterations in nucleic acids or the proteins they encode.According to one embodiment, the ordinary skilled artisan can analyzethe one or more genes for mutations including deletion mutants,insertion mutants, frame shift mutants, nonsense mutants, missensemutant, and splice mutants.

Nucleic acid used for analysis of the one or more genes can be isolatedfrom cells in the sample according to standard methodologies (Sambrooket al., 1989). The nucleic acid, for example, may be genomic DNA orfractionated or whole cell RNA, or miRNA acquired from exosomes or cellsurfaces. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA; in another, it is exosomal RNA. Normally, thenucleic acid is amplified. Depending on the format of the assay foranalyzing the one or more genes, the specific nucleic acid of interestis identified in the sample directly using amplification or with asecond, known nucleic acid following amplification. Next, the identifiedproduct is detected. In certain applications, the detection may beperformed by visual means (e.g., ethidium bromide staining of a gel).Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of radiolabel orfluorescent label or even via a system using electrical or thermalimpulse signals (Affymax Technology; Bellus, 1994).

Various types of defects are known to occur in the biomarkers of theinvention. Alterations include without limitation deletions, insertions,point mutations, and duplications. Point mutations can be silent or canresult in stop codons, frame shift mutations or amino acidsubstitutions. Mutations in and outside the coding region of the one ormore genes may occur and can be analyzed according to the methods of theinvention. The target site of a nucleic acid of interest can include theregion wherein the sequence varies. Examples include, but are notlimited to, polymorphisms which exist in different forms such as singlenucleotide variations, nucleotide repeats, multibase deletion (more thanone nucleotide deleted from the consensus sequence), multibase insertion(more than one nucleotide inserted from the consensus sequence),microsatellite repeats (small numbers of nucleotide repeats with atypical 5-1000 repeat units), di-nucleotide repeats, tri-nucleotiderepeats, sequence rearrangements (including translocation andduplication), chimeric sequence (two sequences from different geneorigins are fused together), and the like. Among sequence polymorphisms,the most frequent polymorphisms in the human genome are single-basevariations, also called single-nucleotide polymorphisms (SNPs). SNPs areabundant, stable and widely distributed across the genome.

Molecular profiling includes methods for haplotyping one or more genes.The haplotype is a set of genetic determinants located on a singlechromosome and it typically contains a particular combination of alleles(all the alternative sequences of a gene) in a region of a chromosome.In other words, the haplotype is phased sequence information onindividual chromosomes. Very often, phased SNPs on a chromosome define ahaplotype. A combination of haplotypes on chromosomes can determine agenetic profile of a cell. It is the haplotype that determines a linkagebetween a specific genetic marker and a disease mutation. Haplotypingcan be done by any methods known in the art. Common methods of scoringSNPs include hybridization microarray or direct gel sequencing, reviewedin Landgren et al., Genome Research, 8:769-776, 1998. For example, onlyone copy of one or more genes can be isolated from an individual and thenucleotide at each of the variant positions is determined.Alternatively, an allele specific PCR or a similar method can be used toamplify only one copy of the one or more genes in an individual, and theSNPs at the variant positions of the present invention are determined.The Clark method known in the art can also be employed for haplotyping.A high throughput molecular haplotyping method is also disclosed in Tostet al., Nucleic Acids Res., 30(19):e96 (2002), which is incorporatedherein by reference.

Thus, additional variant(s) that are in linkage disequilibrium with thevariants and/or haplotypes of the present invention can be identified bya haplotyping method known in the art, as will be apparent to a skilledartisan in the field of genetics and haplotyping. The additionalvariants that are in linkage disequilibrium with a variant or haplotypeof the present invention can also be useful in the various applicationsas described below.

For purposes of genotyping and haplotyping, both genomic DNA andmRNA/cDNA can be used, and both are herein referred to generically as“gene.”

Numerous techniques for detecting nucleotide variants are known in theart and can all be used for the method of this invention. The techniquescan be protein-based or nucleic acid-based. In either case, thetechniques used must be sufficiently sensitive so as to accuratelydetect the small nucleotide or amino acid variations. Very often, aprobe is used which is labeled with a detectable marker. Unlessotherwise specified in a particular technique described below, anysuitable marker known in the art can be used, including but not limitedto, radioactive isotopes, fluorescent compounds, biotin which isdetectable using streptavidin, enzymes (e.g., alkaline phosphatase),substrates of an enzyme, ligands and antibodies, etc. See Jablonski etal., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al.,Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol. Biol.,113:237-251 (1977).

In a nucleic acid-based detection method, target DNA sample, i.e., asample containing genomic DNA, cDNA, mRNA and/or miRNA, corresponding tothe one or more genes must be obtained from the individual to be tested.Any tissue or cell sample containing the genomic DNA, miRNA, mRNA,and/or cDNA (or a portion thereof) corresponding to the one or moregenes can be used. For this purpose, a tissue sample containing cellnucleus and thus genomic DNA can be obtained from the individual. Bloodsamples can also be useful except that only white blood cells and otherlymphocytes have cell nucleus, while red blood cells are without anucleus and contain only mRNA or miRNA. Nevertheless, miRNA and mRNA arealso useful as either can be analyzed for the presence of nucleotidevariants in its sequence or serve as template for cDNA synthesis. Thetissue or cell samples can be analyzed directly without much processing.Alternatively, nucleic acids including the target sequence can beextracted, purified, and/or amplified before they are subject to thevarious detecting procedures discussed below. Other than tissue or cellsamples, cDNAs or genomic DNAs from a cDNA or genomic DNA libraryconstructed using a tissue or cell sample obtained from the individualto be tested are also useful.

To determine the presence or absence of a particular nucleotide variant,sequencing of the target genomic DNA or cDNA, particularly the regionencompassing the nucleotide variant locus to be detected. Varioussequencing techniques are generally known and widely used in the artincluding the Sanger method and Gilbert chemical method. Thepyrosequencing method monitors DNA synthesis in real time using aluminometric detection system. Pyrosequencing has been shown to beeffective in analyzing genetic polymorphisms such as single-nucleotidepolymorphisms and can also be used in the present invention. SeeNordstrom et al., Biotechnol. Appl. Biochem., 31(2):107-112 (2000);Ahmadian et al., Anal. Biochem., 280:103-110 (2000).

Nucleic acid variants can be detected by a suitable detection process.Non limiting examples of methods of detection, quantification,sequencing and the like are; mass detection of mass modified amplicons(e.g., matrix-assisted laser desorption ionization (MALDI) massspectrometry and electrospray (ES) mass spectrometry), a primerextension method (e.g., iPLEX™; Sequenom, Inc.), microsequencing methods(e.g., a modification of primer extension methodology), ligase sequencedetermination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, andWO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat.Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), direct DNAsequencing, restriction fragment length polymorphism (RFLP analysis),allele specific oligonucleotide (ASO) analysis, methylation-specific PCR(MSPCR), pyrosequencing analysis (see above), acycloprime analysis,Reverse dot blot, GeneChip microarrays, Dynamic allele-specifichybridization (DASH), Peptide nucleic acid (PNA) and locked nucleicacids (LNA) probes, TaqMan, Molecular Beacons, Intercalating dye, FRETprimers, AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplexminisequencing, SNaPshot, GOOD assay, Microarray miniseq, arrayed primerextension (APEX), Microarray primer extension (e.g., microarray sequencedetermination methods), Tag arrays, Coded microspheres,Template-directed incorporation (TDI), fluorescence polarization,Colorimetric oligonucleotide ligation assay (OLA), Sequence-coded OLA,Microarray ligation, Ligase chain reaction, Padlock probes, Invaderassay, hybridization methods (e.g., hybridization using at least oneprobe, hybridization using at least one fluorescently labeled probe, andthe like), conventional dot blot analyses, single strand conformationalpolymorphism analysis (SSCP, e.g., U.S. Pat. Nos. 5,891,625 and6,013,499; Orita et al., Proc. Natl. Acad. Sci. U.S.A. 86: 27776-2770(1989)), denaturing gradient gel electrophoresis (DGGE), heteroduplexanalysis, mismatch cleavage detection, and techniques described inSheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706 (1991), Whiteet al., Genomics 12: 301-306 (1992), Grompe et al., Proc. Natl. Acad.Sci. USA 86: 5855-5892 (1989), and Grompe, Nature Genetics 5: 111-117(1993), cloning and sequencing, electrophoresis, the use ofhybridization probes and quantitative real time polymerase chainreaction (QRT-PCR), digital PCR, nanopore sequencing, chips andcombinations thereof. The detection and quantification of alleles orparalogs can be carried out using the “closed-tube” methods described inU.S. patent application Ser. No. 11/950,395, filed on Dec. 4, 2007. Insome embodiments the amount of a nucleic acid species is determined bymass spectrometry, primer extension, sequencing (e.g., any suitablemethod, for example nanopore or pyrosequencing), Quantitative PCR (Q-PCRor QRT-PCR), digital PCR, combinations thereof, and the like.

The term “sequence analysis” as used herein refers to determining anucleotide sequence, e.g., that of an amplification product. The entiresequence or a partial sequence of a polynucleotide, e.g., DNA or mRNA,can be determined, and the determined nucleotide sequence can bereferred to as a “read” or “sequence read.” For example, linearamplification products may be analyzed directly without furtheramplification in some embodiments (e.g., by using single-moleculesequencing methodology). In certain embodiments, linear amplificationproducts may be subject to further amplification and then analyzed(e.g., using sequencing by ligation or pyrosequencing methodology).Reads may be subject to different types of sequence analysis. Anysuitable sequencing method can be used to detect, and determine theamount of, nucleotide sequence species, amplified nucleic acid species,or detectable products generated from the foregoing. Examples of certainsequencing methods are described hereafter.

A sequence analysis apparatus or sequence analysis component(s) includesan apparatus, and one or more components used in conjunction with suchapparatus, that can be used by a person of ordinary skill to determine anucleotide sequence resulting from processes described herein (e.g.,linear and/or exponential amplification products). Examples ofsequencing platforms include, without limitation, the 454 platform(Roche) (Margulies, M. et al. 2005 Nature 437, 376-380), IlluminaGenomic Analyzer (or Solexa platform) or SOLID System (AppliedBiosystems; see PCT patent application publications WO 06/084132entitled “Reagents, Methods, and Libraries For Bead-Based Sequencing”and WO07/121,489 entitled “Reagents, Methods, and Libraries for Gel-FreeBead-Based Sequencing.”) or the Helicos True Single Molecule DNAsequencing technology (Harris T D et al. 2008 Science, 320, 106-109),the single molecule, real-time (SMRT™) technology of PacificBiosciences, nanopore sequencing (Soni G V and Meller A. 2007 Clin Chem53: 1996-2001), Ion semiconductor sequencing (Ion Torrent Systems, Inc,San Francisco, Calif.), or DNA nanoball sequencing (Complete Genomics,Mountain View, Calif.), VisiGen Biotechnologies approach (Invitrogen)and polony sequencing. Such platforms allow sequencing of many nucleicacid molecules isolated from a specimen at high orders of multiplexingin a parallel manner (Dear, Brief Funct Genomic Proteomic 2003; 1:397-416; Haimovich, Methods, challenges, and promise of next-generationsequencing in cancer biology. Yale J Biol Med. 2011 December;84(4):439-46). These non-Sanger-based sequencing technologies aresometimes referred to as NextGen sequencing, NGS, next-generationsequencing, next generation sequencing, and variations thereof.Typically they allow much higher throughput than the traditional Sangerapproach. See Schuster, Next-generation sequencing transforms today'sbiology, Nature Methods 5:16-18 (2008); Metzker, Sequencingtechnologies—the next generation. Nat Rev Genet. 2010 January;11(1):31-46. These platforms can allow sequencing of clonally expandedor non-amplified single molecules of nucleic acid fragments. Certainplatforms involve, for example, sequencing by ligation of dye-modifiedprobes (including cyclic ligation and cleavage), pyrosequencing, andsingle-molecule sequencing. Nucleotide sequence species, amplificationnucleic acid species and detectable products generated there from can beanalyzed by such sequence analysis platforms. Next-generation sequencingcan be used in the methods of the invention, e.g., to determinemutations, copy number, or expression levels, as appropriate. Themethods can be used to perform whole genome sequencing or sequencing ofspecific sequences of interest, such as a gene of interest or a fragmentthereof.

Sequencing by ligation is a nucleic acid sequencing method that relieson the sensitivity of DNA ligase to base-pairing mismatch. DNA ligasejoins together ends of DNA that are correctly base paired. Combining theability of DNA ligase to join together only correctly base paired DNAends, with mixed pools of fluorescently labeled oligonucleotides orprimers, enables sequence determination by fluorescence detection.Longer sequence reads may be obtained by including primers containingcleavable linkages that can be cleaved after label identification.Cleavage at the linker removes the label and regenerates the 5′phosphate on the end of the ligated primer, preparing the primer foranother round of ligation. In some embodiments primers may be labeledwith more than one fluorescent label, e.g., at least 1, 2, 3, 4, or 5fluorescent labels.

Sequencing by ligation generally involves the following steps. Clonalbead populations can be prepared in emulsion microreactors containingtarget nucleic acid template sequences, amplification reactioncomponents, beads and primers. After amplification, templates aredenatured and bead enrichment is performed to separate beads withextended templates from undesired beads (e.g., beads with no extendedtemplates). The template on the selected beads undergoes a 3′modification to allow covalent bonding to the slide, and modified beadscan be deposited onto a glass slide. Deposition chambers offer theability to segment a slide into one, four or eight chambers during thebead loading process. For sequence analysis, primers hybridize to theadapter sequence. A set of four color dye-labeled probes competes forligation to the sequencing primer. Specificity of probe ligation isachieved by interrogating every 4th and 5th base during the ligationseries. Five to seven rounds of ligation, detection and cleavage recordthe color at every 5th position with the number of rounds determined bythe type of library used. Following each round of ligation, a newcomplimentary primer offset by one base in the 5′ direction is laid downfor another series of ligations. Primer reset and ligation rounds (5-7ligation cycles per round) are repeated sequentially five times togenerate 25-35 base pairs of sequence for a single tag. With mate-pairedsequencing, this process is repeated for a second tag.

Pyrosequencing is a nucleic acid sequencing method based on sequencingby synthesis, which relies on detection of a pyrophosphate released onnucleotide incorporation. Generally, sequencing by synthesis involvessynthesizing, one nucleotide at a time, a DNA strand complimentary tothe strand whose sequence is being sought. Target nucleic acids may beimmobilized to a solid support, hybridized with a sequencing primer,incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. Nucleotide solutions aresequentially added and removed. Correct incorporation of a nucleotidereleases a pyrophosphate, which interacts with ATP sulfurylase andproduces ATP in the presence of adenosine 5′ phosphosulfate, fueling theluciferin reaction, which produces a chemiluminescent signal allowingsequence determination. The amount of light generated is proportional tothe number of bases added. Accordingly, the sequence downstream of thesequencing primer can be determined. An illustrative system forpyrosequencing involves the following steps: ligating an adaptor nucleicacid to a nucleic acid under investigation and hybridizing the resultingnucleic acid to a bead; amplifying a nucleotide sequence in an emulsion;sorting beads using a picoliter multiwell solid support; and sequencingamplified nucleotide sequences by pyrosequencing methodology (e.g.,Nakano et al., “Single-molecule PCR using water-in-oil emulsion;”Journal of Biotechnology 102: 117-124 (2003)).

Certain single-molecule sequencing embodiments are based on theprincipal of sequencing by synthesis, and use single-pair FluorescenceResonance Energy Transfer (single pair FRET) as a mechanism by whichphotons are emitted as a result of successful nucleotide incorporation.The emitted photons often are detected using intensified or highsensitivity cooled charge-couple-devices in conjunction with totalinternal reflection microscopy (TIRM). Photons are only emitted when theintroduced reaction solution contains the correct nucleotide forincorporation into the growing nucleic acid chain that is synthesized asa result of the sequencing process. In FRET based single-moleculesequencing, energy is transferred between two fluorescent dyes,sometimes polymethine cyanine dyes Cy3 and Cy5, through long-rangedipole interactions. The donor is excited at its specific excitationwavelength and the excited state energy is transferred, non-radiativelyto the acceptor dye, which in turn becomes excited. The acceptor dyeeventually returns to the ground state by radiative emission of aphoton. The two dyes used in the energy transfer process represent the“single pair” in single pair FRET. Cy3 often is used as the donorfluorophore and often is incorporated as the first labeled nucleotide.Cy5 often is used as the acceptor fluorophore and is used as thenucleotide label for successive nucleotide additions after incorporationof a first Cy3 labeled nucleotide. The fluorophores generally are within10 nanometers of each for energy transfer to occur successfully.

An example of a system that can be used based on single-moleculesequencing generally involves hybridizing a primer to a target nucleicacid sequence to generate a complex; associating the complex with asolid phase; iteratively extending the primer by a nucleotide taggedwith a fluorescent molecule; and capturing an image of fluorescenceresonance energy transfer signals after each iteration (e.g., U.S. Pat.No. 7,169,314; Braslaysky et al., PNAS 100(7): 3960-3964 (2003)). Such asystem can be used to directly sequence amplification products (linearlyor exponentially amplified products) generated by processes describedherein. In some embodiments the amplification products can be hybridizedto a primer that contains sequences complementary to immobilized capturesequences present on a solid support, a bead or glass slide for example.Hybridization of the primer-amplification product complexes with theimmobilized capture sequences, immobilizes amplification products tosolid supports for single pair FRET based sequencing by synthesis. Theprimer often is fluorescent, so that an initial reference image of thesurface of the slide with immobilized nucleic acids can be generated.The initial reference image is useful for determining locations at whichtrue nucleotide incorporation is occurring. Fluorescence signalsdetected in array locations not initially identified in the “primeronly” reference image are discarded as non-specific fluorescence.Following immobilization of the primer-amplification product complexes,the bound nucleic acids often are sequenced in parallel by the iterativesteps of, a) polymerase extension in the presence of one fluorescentlylabeled nucleotide, b) detection of fluorescence using appropriatemicroscopy, TIRM for example, c) removal of fluorescent nucleotide, andd) return to step a with a different fluorescently labeled nucleotide.

In some embodiments, nucleotide sequencing may be by solid phase singlenucleotide sequencing methods and processes. Solid phase singlenucleotide sequencing methods involve contacting target nucleic acid andsolid support under conditions in which a single molecule of samplenucleic acid hybridizes to a single molecule of a solid support. Suchconditions can include providing the solid support molecules and asingle molecule of target nucleic acid in a “microreactor.” Suchconditions also can include providing a mixture in which the targetnucleic acid molecule can hybridize to solid phase nucleic acid on thesolid support. Single nucleotide sequencing methods useful in theembodiments described herein are described in U.S. Provisional PatentApplication Ser. No. 61/021,871 filed Jan. 17, 2008.

In certain embodiments, nanopore sequencing detection methods include(a) contacting a target nucleic acid for sequencing (“base nucleicacid,” e.g., linked probe molecule) with sequence-specific detectors,under conditions in which the detectors specifically hybridize tosubstantially complementary subsequences of the base nucleic acid; (b)detecting signals from the detectors and (c) determining the sequence ofthe base nucleic acid according to the signals detected. In certainembodiments, the detectors hybridized to the base nucleic acid aredisassociated from the base nucleic acid (e.g., sequentiallydissociated) when the detectors interfere with a nanopore structure asthe base nucleic acid passes through a pore, and the detectorsdisassociated from the base sequence are detected. In some embodiments,a detector disassociated from a base nucleic acid emits a detectablesignal, and the detector hybridized to the base nucleic acid emits adifferent detectable signal or no detectable signal. In certainembodiments, nucleotides in a nucleic acid (e.g., linked probe molecule)are substituted with specific nucleotide sequences corresponding tospecific nucleotides (“nucleotide representatives”), thereby giving riseto an expanded nucleic acid (e.g., U.S. Pat. No. 6,723,513), and thedetectors hybridize to the nucleotide representatives in the expandednucleic acid, which serves as a base nucleic acid. In such embodiments,nucleotide representatives may be arranged in a binary or higher orderarrangement (e.g., Soni and Meller, Clinical Chemistry 53(11): 1996-2001(2007)). In some embodiments, a nucleic acid is not expanded, does notgive rise to an expanded nucleic acid, and directly serves a basenucleic acid (e.g., a linked probe molecule serves as a non-expandedbase nucleic acid), and detectors are directly contacted with the basenucleic acid. For example, a first detector may hybridize to a firstsubsequence and a second detector may hybridize to a second subsequence,where the first detector and second detector each have detectable labelsthat can be distinguished from one another, and where the signals fromthe first detector and second detector can be distinguished from oneanother when the detectors are disassociated from the base nucleic acid.In certain embodiments, detectors include a region that hybridizes tothe base nucleic acid (e.g., two regions), which can be about 3 to about100 nucleotides in length (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,85, 90, or 95 nucleotides in length). A detector also may include one ormore regions of nucleotides that do not hybridize to the base nucleicacid. In some embodiments, a detector is a molecular beacon. A detectoroften comprises one or more detectable labels independently selectedfrom those described herein. Each detectable label can be detected byany convenient detection process capable of detecting a signal generatedby each label (e.g., magnetic, electric, chemical, optical and thelike). For example, a CD camera can be used to detect signals from oneor more distinguishable quantum dots linked to a detector.

In certain sequence analysis embodiments, reads may be used to constructa larger nucleotide sequence, which can be facilitated by identifyingoverlapping sequences in different reads and by using identificationsequences in the reads. Such sequence analysis methods and software forconstructing larger sequences from reads are known to the person ofordinary skill (e.g., Venter et al., Science 291: 1304-1351 (2001)).Specific reads, partial nucleotide sequence constructs, and fullnucleotide sequence constructs may be compared between nucleotidesequences within a sample nucleic acid (i.e., internal comparison) ormay be compared with a reference sequence (i.e., reference comparison)in certain sequence analysis embodiments. Internal comparisons can beperformed in situations where a sample nucleic acid is prepared frommultiple samples or from a single sample source that contains sequencevariations. Reference comparisons sometimes are performed when areference nucleotide sequence is known and an objective is to determinewhether a sample nucleic acid contains a nucleotide sequence that issubstantially similar or the same, or different, than a referencenucleotide sequence. Sequence analysis can be facilitated by the use ofsequence analysis apparatus and components described above.

Primer extension polymorphism detection methods, also referred to hereinas “microsequencing” methods, typically are carried out by hybridizing acomplementary oligonucleotide to a nucleic acid carrying the polymorphicsite. In these methods, the oligonucleotide typically hybridizesadjacent to the polymorphic site. The term “adjacent” as used inreference to “microsequencing” methods, refers to the 3′ end of theextension oligonucleotide being sometimes 1 nucleotide from the 5′ endof the polymorphic site, often 2 or 3, and at times 4, 5, 6, 7, 8, 9, or10 nucleotides from the 5′ end of the polymorphic site, in the nucleicacid when the extension oligonucleotide is hybridized to the nucleicacid. The extension oligonucleotide then is extended by one or morenucleotides, often 1, 2, or 3 nucleotides, and the number and/or type ofnucleotides that are added to the extension oligonucleotide determinewhich polymorphic variant or variants are present. Oligonucleotideextension methods are disclosed, for example, in U.S. Pat. Nos.4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755;5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702;6,046,005; 6,087,095; 6,210,891; and WO 01/20039. The extension productscan be detected in any manner, such as by fluorescence methods (see,e.g., Chen & Kwok, Nucleic Acids Research 25: 347-353 (1997) and Chen etal., Proc. Natl. Acad. Sci. USA 94/20: 10756-10761 (1997)) or by massspectrometric methods (e.g., MALDI-TOF mass spectrometry) and othermethods described herein. Oligonucleotide extension methods using massspectrometry are described, for example, in U.S. Pat. Nos. 5,547,835;5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031;6,194,144; and 6,258,538.

Microsequencing detection methods often incorporate an amplificationprocess that proceeds the extension step. The amplification processtypically amplifies a region from a nucleic acid sample that comprisesthe polymorphic site. Amplification can be carried out using methodsdescribed above, or for example using a pair of oligonucleotide primersin a polymerase chain reaction (PCR), in which one oligonucleotideprimer typically is complementary to a region 3′ of the polymorphism andthe other typically is complementary to a region 5′ of the polymorphism.A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos.4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO01/27327; and WO 01/27329 for example. PCR primer pairs may also be usedin any commercially available machines that perform PCR, such as any ofthe GeneAmp™ Systems available from Applied Biosystems.

Other appropriate sequencing methods include multiplex polony sequencing(as described in Shendure et al., Accurate Multiplex Polony Sequencingof an Evolved Bacterial Genome, Sciencexpress, Aug. 4, 2005, pg 1available at www.sciencexpress.org/4 Aug.2005/Page1/10.1126/science.1117389, incorporated herein by reference),which employs immobilized microbeads, and sequencing in microfabricatedpicoliter reactors (as described in Margulies et al., Genome Sequencingin Microfabricated High-Density Picolitre Reactors, Nature, August 2005,available at www.nature.com/nature (published online 31 Jul. 2005,doi:10.1038/nature03959, incorporated herein by reference).

Whole genome sequencing may also be used for discriminating alleles ofRNA transcripts, in some embodiments. Examples of whole genomesequencing methods include, but are not limited to, nanopore-basedsequencing methods, sequencing by synthesis and sequencing by ligation,as described above.

Nucleic acid variants can also be detected using standardelectrophoretic techniques. Although the detection step can sometimes bepreceded by an amplification step, amplification is not required in theembodiments described herein. Examples of methods for detection andquantification of a nucleic acid using electrophoretic techniques can befound in the art. A non-limiting example comprises running a sample(e.g., mixed nucleic acid sample isolated from maternal serum, oramplification nucleic acid species, for example) in an agarose orpolyacrylamide gel. The gel may be labeled (e.g., stained) with ethidiumbromide (see, Sambrook and Russell, Molecular Cloning: A LaboratoryManual 3d ed., 2001). The presence of a band of the same size as thestandard control is an indication of the presence of a target nucleicacid sequence, the amount of which may then be compared to the controlbased on the intensity of the band, thus detecting and quantifying thetarget sequence of interest. In some embodiments, restriction enzymescapable of distinguishing between maternal and paternal alleles may beused to detect and quantify target nucleic acid species. In certainembodiments, oligonucleotide probes specific to a sequence of interestare used to detect the presence of the target sequence of interest. Theoligonucleotides can also be used to indicate the amount of the targetnucleic acid molecules in comparison to the standard control, based onthe intensity of signal imparted by the probe.

Sequence-specific probe hybridization can be used to detect a particularnucleic acid in a mixture or mixed population comprising other speciesof nucleic acids. Under sufficiently stringent hybridization conditions,the probes hybridize specifically only to substantially complementarysequences. The stringency of the hybridization conditions can be relaxedto tolerate varying amounts of sequence mismatch. A number ofhybridization formats are known in the art, which include but are notlimited to, solution phase, solid phase, or mixed phase hybridizationassays. The following articles provide an overview of the varioushybridization assay formats: Singer et al., Biotechniques 4:230, 1986;Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson, In situHybridization, Wilkinson ed., IRL Press, Oxford University Press,Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: APractical Approach, IRL Press, 1987.

Hybridization complexes can be detected by techniques known in the art.Nucleic acid probes capable of specifically hybridizing to a targetnucleic acid (e.g., mRNA or DNA) can be labeled by any suitable method,and the labeled probe used to detect the presence of hybridized nucleicacids. One commonly used method of detection is autoradiography, usingprobes labeled with ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, or the like. Thechoice of radioactive isotope depends on research preferences due toease of synthesis, stability, and half-lives of the selected isotopes.Other labels include compounds (e.g., biotin and digoxigenin), whichbind to antiligands or antibodies labeled with fluorophores,chemiluminescent agents, and enzymes. In some embodiments, probes can beconjugated directly with labels such as fluorophores, chemiluminescentagents or enzymes. The choice of label depends on sensitivity required,ease of conjugation with the probe, stability requirements, andavailable instrumentation.

Alternatively, the restriction fragment length polymorphism (RFLP) andAFLP method may be used for molecular profiling. If a nucleotide variantin the target DNA corresponding to the one or more genes results in theelimination or creation of a restriction enzyme recognition site, thendigestion of the target DNA with that particular restriction enzyme willgenerate an altered restriction fragment length pattern. Thus, adetected RFLP or AFLP will indicate the presence of a particularnucleotide variant.

Another useful approach is the single-stranded conformation polymorphismassay (SSCA), which is based on the altered mobility of asingle-stranded target DNA spanning the nucleotide variant of interest.A single nucleotide change in the target sequence can result indifferent intramolecular base pairing pattern, and thus differentsecondary structure of the single-stranded DNA, which can be detected ina non-denaturing gel. See Orita et al., Proc. Natl. Acad. Sci. USA,86:2776-2770 (1989). Denaturing gel-based techniques such as clampeddenaturing gel electrophoresis (CDGE) and denaturing gradient gelelectrophoresis (DGGE) detect differences in migration rates of mutantsequences as compared to wild-type sequences in denaturing gel. SeeMiller et al., Biotechniques, 5:1016-24 (1999); Sheffield et al., Am. J.Hum, Genet., 49:699-706 (1991); Wartell et al., Nucleic Acids Res.,18:2699-2705 (1990); and Sheffield et al., Proc. Natl. Acad. Sci. USA,86:232-236 (1989). In addition, the double-strand conformation analysis(DSCA) can also be useful in the present invention. See Arguello et al.,Nat. Genet., 18:192-194 (1998).

The presence or absence of a nucleotide variant at a particular locus inthe one or more genes of an individual can also be detected using theamplification refractory mutation system (ARMS) technique. See e.g.,European Patent No. 0,332,435; Newton et al., Nucleic Acids Res.,17:2503-2515 (1989); Fox et al., Br. J. Cancer, 77:1267-1274 (1998);Robertson et al., Eur. Respir. J., 12:477-482 (1998). In the ARMSmethod, a primer is synthesized matching the nucleotide sequenceimmediately 5′ upstream from the locus being tested except that the3′-end nucleotide which corresponds to the nucleotide at the locus is apredetermined nucleotide. For example, the 3′-end nucleotide can be thesame as that in the mutated locus. The primer can be of any suitablelength so long as it hybridizes to the target DNA under stringentconditions only when its 3′-end nucleotide matches the nucleotide at thelocus being tested. Preferably the primer has at least 12 nucleotides,more preferably from about 18 to 50 nucleotides. If the individualtested has a mutation at the locus and the nucleotide therein matchesthe 3′-end nucleotide of the primer, then the primer can be furtherextended upon hybridizing to the target DNA template, and the primer caninitiate a PCR amplification reaction in conjunction with anothersuitable PCR primer. In contrast, if the nucleotide at the locus is ofwild type, then primer extension cannot be achieved. Various forms ofARMS techniques developed in the past few years can be used. See e.g.,Gibson et al., Clin. Chem. 43:1336-1341 (1997).

Similar to the ARMS technique is the mini sequencing or singlenucleotide primer extension method, which is based on the incorporationof a single nucleotide. An oligonucleotide primer matching thenucleotide sequence immediately 5′ to the locus being tested ishybridized to the target DNA, mRNA or miRNA in the presence of labeleddideoxyribonucleotides. A labeled nucleotide is incorporated or linkedto the primer only when the dideoxyribonucleotides matches thenucleotide at the variant locus being detected. Thus, the identity ofthe nucleotide at the variant locus can be revealed based on thedetection label attached to the incorporated dideoxyribonucleotides. SeeSyvanen et al., Genomics, 8:684-692 (1990); Shumaker et al., Hum.Mutat., 7:346-354 (1996); Chen et al., Genome Res., 10:549-547 (2000).

Another set of techniques useful in the present invention is theso-called “oligonucleotide ligation assay” (OLA) in whichdifferentiation between a wild-type locus and a mutation is based on theability of two oligonucleotides to anneal adjacent to each other on thetarget DNA molecule allowing the two oligonucleotides joined together bya DNA ligase. See Landergren et al., Science, 241:1077-1080 (1988); Chenet al, Genome Res., 8:549-556 (1998); Iannone et al., Cytometry,39:131-140 (2000). Thus, for example, to detect a single-nucleotidemutation at a particular locus in the one or more genes, twooligonucleotides can be synthesized, one having the sequence just 5′upstream from the locus with its 3′ end nucleotide being identical tothe nucleotide in the variant locus of the particular gene, the otherhaving a nucleotide sequence matching the sequence immediately 3′downstream from the locus in the gene. The oligonucleotides can belabeled for the purpose of detection. Upon hybridizing to the targetgene under a stringent condition, the two oligonucleotides are subjectto ligation in the presence of a suitable ligase. The ligation of thetwo oligonucleotides would indicate that the target DNA has a nucleotidevariant at the locus being detected.

Detection of small genetic variations can also be accomplished by avariety of hybridization-based approaches. Allele-specificoligonucleotides are most useful. See Conner et al., Proc. Natl. Acad.Sci. USA, 80:278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA,86:6230-6234 (1989). Oligonucleotide probes (allele-specific)hybridizing specifically to a gene allele having a particular genevariant at a particular locus but not to other alleles can be designedby methods known in the art. The probes can have a length of, e.g., from10 to about 50 nucleotide bases. The target DNA and the oligonucleotideprobe can be contacted with each other under conditions sufficientlystringent such that the nucleotide variant can be distinguished from thewild-type gene based on the presence or absence of hybridization. Theprobe can be labeled to provide detection signals. Alternatively, theallele-specific oligonucleotide probe can be used as a PCR amplificationprimer in an “allele-specific PCR” and the presence or absence of a PCRproduct of the expected length would indicate the presence or absence ofa particular nucleotide variant.

Other useful hybridization-based techniques allow two single-strandednucleic acids annealed together even in the presence of mismatch due tonucleotide substitution, insertion or deletion. The mismatch can then bedetected using various techniques. For example, the annealed duplexescan be subject to electrophoresis. The mismatched duplexes can bedetected based on their electrophoretic mobility that is different fromthe perfectly matched duplexes. See Cariello, Human Genetics, 42:726(1988). Alternatively, in an RNase protection assay, a RNA probe can beprepared spanning the nucleotide variant site to be detected and havinga detection marker. See Giunta et al., Diagn. Mol. Path., 5:265-270(1996); Finkelstein et al., Genomics, 7:167-172 (1990); Kinszler et al.,Science 251:1366-1370 (1991). The RNA probe can be hybridized to thetarget DNA or mRNA forming a heteroduplex that is then subject to theribonuclease RNase A digestion. RNase A digests the RNA probe in theheteroduplex only at the site of mismatch. The digestion can bedetermined on a denaturing electrophoresis gel based on size variations.In addition, mismatches can also be detected by chemical cleavagemethods known in the art. See e.g., Roberts et al., Nucleic Acids Res.,25:3377-3378 (1997).

In the mutS assay, a probe can be prepared matching the gene sequencesurrounding the locus at which the presence or absence of a mutation isto be detected, except that a predetermined nucleotide is used at thevariant locus. Upon annealing the probe to the target DNA to form aduplex, the E. coli mutS protein is contacted with the duplex. Since themutS protein binds only to heteroduplex sequences containing anucleotide mismatch, the binding of the mutS protein will be indicativeof the presence of a mutation. See Modrich et al., Ann. Rev. Genet.,25:229-253 (1991).

A great variety of improvements and variations have been developed inthe art on the basis of the above-described basic techniques which canbe useful in detecting mutations or nucleotide variants in the presentinvention. For example, the “sunrise probes” or “molecular beacons” usethe fluorescence resonance energy transfer (FRET) property and give riseto high sensitivity. See Wolf et al., Proc. Nat. Acad. Sci. USA,85:8790-8794 (1988). Typically, a probe spanning the nucleotide locus tobe detected are designed into a hairpin-shaped structure and labeledwith a quenching fluorophore at one end and a reporter fluorophore atthe other end. In its natural state, the fluorescence from the reporterfluorophore is quenched by the quenching fluorophore due to theproximity of one fluorophore to the other. Upon hybridization of theprobe to the target DNA, the 5′ end is separated apart from the 3′-endand thus fluorescence signal is regenerated. See Nazarenko et al.,Nucleic Acids Res., 25:2516-2521 (1997); Rychlik et al., Nucleic AcidsRes., 17:8543-8551 (1989); Sharkey et al., Bio/Technology 12:506-509(1994); Tyagi et al., Nat. Biotechnol., 14:303-308 (1996); Tyagi et al.,Nat. Biotechnol., 16:49-53 (1998). The homo-tag assisted non-dimersystem (HANDS) can be used in combination with the molecular beaconmethods to suppress primer-dimer accumulation. See Brownie et al.,Nucleic Acids Res., 25:3235-3241 (1997).

Dye-labeled oligonucleotide ligation assay is a FRET-based method, whichcombines the OLA assay and PCR. See Chen et al., Genome Res. 8:549-556(1998). TaqMan is another FRET-based method for detecting nucleotidevariants. A TaqMan probe can be oligonucleotides designed to have thenucleotide sequence of the gene spanning the variant locus of interestand to differentially hybridize with different alleles. The two ends ofthe probe are labeled with a quenching fluorophore and a reporterfluorophore, respectively. The TaqMan probe is incorporated into a PCRreaction for the amplification of a target gene region containing thelocus of interest using Taq polymerase. As Taq polymerase exhibits 5′-3′exonuclease activity but has no 3′-5′ exonuclease activity, if theTaqMan probe is annealed to the target DNA template, the 5′-end of theTaqMan probe will be degraded by Taq polymerase during the PCR reactionthus separating the reporting fluorophore from the quenching fluorophoreand releasing fluorescence signals. See Holland et al., Proc. Natl.Acad. Sci. USA, 88:7276-7280 (1991); Kalinina et al., Nucleic AcidsRes., 25:1999-2004 (1997); Whitcombe et al., Clin. Chem., 44:918-923(1998).

In addition, the detection in the present invention can also employ achemiluminescence-based technique. For example, an oligonucleotide probecan be designed to hybridize to either the wild-type or a variant genelocus but not both. The probe is labeled with a highly chemiluminescentacridinium ester. Hydrolysis of the acridinium ester destroyschemiluminescence. The hybridization of the probe to the target DNAprevents the hydrolysis of the acridinium ester. Therefore, the presenceor absence of a particular mutation in the target DNA is determined bymeasuring chemiluminescence changes. See Nelson et al., Nucleic AcidsRes., 24:4998-5003 (1996).

The detection of genetic variation in the gene in accordance with thepresent invention can also be based on the “base excision sequencescanning” (BESS) technique. The BESS method is a PCR-based mutationscanning method. BESS T-Scan and BESS G-Tracker are generated which areanalogous to T and G ladders of dideoxy sequencing. Mutations aredetected by comparing the sequence of normal and mutant DNA. See, e.g.,Hawkins et al., Electrophoresis, 20:1171-1176 (1999).

Mass spectrometry can be used for molecular profiling according to theinvention. See Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998).For example, in the primer oligo base extension (PROBE™) method, atarget nucleic acid is immobilized to a solid-phase support. A primer isannealed to the target immediately 5′ upstream from the locus to beanalyzed. Primer extension is carried out in the presence of a selectedmixture of deoxyribonucleotides and dideoxyribonucleotides. Theresulting mixture of newly extended primers is then analyzed byMALDI-TOF. See e.g., Monforte et al., Nat. Med., 3:360-362 (1997).

In addition, the microchip or microarray technologies are alsoapplicable to the detection method of the present invention.Essentially, in microchips, a large number of different oligonucleotideprobes are immobilized in an array on a substrate or carrier, e.g., asilicon chip or glass slide. Target nucleic acid sequences to beanalyzed can be contacted with the immobilized oligonucleotide probes onthe microchip. See Lipshutz et al., Biotechniques, 19:442-447 (1995);Chee et al., Science, 274:610-614 (1996); Kozal et al., Nat. Med.2:753-759 (1996); Hacia et al., Nat. Genet., 14:441-447 (1996); Saiki etal., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989); Gingeras et al.,Genome Res., 8:435-448 (1998). Alternatively, the multiple targetnucleic acid sequences to be studied are fixed onto a substrate and anarray of probes is contacted with the immobilized target sequences. SeeDrmanac et al., Nat. Biotechnol., 16:54-58 (1998). Numerous microchiptechnologies have been developed incorporating one or more of the abovedescribed techniques for detecting mutations. The microchip technologiescombined with computerized analysis tools allow fast screening in alarge scale. The adaptation of the microchip technologies to the presentinvention will be apparent to a person of skill in the art apprised ofthe present disclosure. See, e.g., U.S. Pat. No. 5,925,525 to Fodor etal; Wilgenbus et al., J. Mol. Med., 77:761-786 (1999); Graber et al.,Curr. Opin. Biotechnol., 9:14-18 (1998); Hacia et al., Nat. Genet.,14:441-447 (1996); Shoemaker et al., Nat. Genet., 14:450-456 (1996);DeRisi et al., Nat. Genet., 14:457-460 (1996); Chee et al., Nat. Genet.,14:610-614 (1996); Lockhart et al., Nat. Genet., 14:675-680 (1996);Drobyshev et al., Gene, 188:45-52 (1997).

As is apparent from the above survey of the suitable detectiontechniques, it may or may not be necessary to amplify the target DNA,i.e., the gene, cDNA, mRNA, miRNA, or a portion thereof to increase thenumber of target DNA molecule, depending on the detection techniquesused. For example, most PCR-based techniques combine the amplificationof a portion of the target and the detection of the mutations. PCRamplification is well known in the art and is disclosed in U.S. Pat.Nos. 4,683,195 and 4,800,159, both which are incorporated herein byreference. For non-PCR-based detection techniques, if necessary, theamplification can be achieved by, e.g., in vivo plasmid multiplication,or by purifying the target DNA from a large amount of tissue or cellsamples. See generally, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989. However, even with scarce samples, many sensitive techniqueshave been developed in which small genetic variations such assingle-nucleotide substitutions can be detected without having toamplify the target DNA in the sample. For example, techniques have beendeveloped that amplify the signal as opposed to the target DNA by, e.g.,employing branched DNA or dendrimers that can hybridize to the targetDNA. The branched or dendrimer DNAs provide multiple hybridization sitesfor hybridization probes to attach thereto thus amplifying the detectionsignals. See Detmer et al., J. Clin. Microbiol., 34:901-907 (1996);Collins et al., Nucleic Acids Res., 25:2979-2984 (1997); Horn et al.,Nucleic Acids Res., 25:4835-4841 (1997); Horn et al., Nucleic AcidsRes., 25:4842-4849 (1997); Nilsen et al., J. Theor. Biol., 187:273-284(1997).

The Invader™ assay is another technique for detecting single nucleotidevariations that can be used for molecular profiling according to theinvention. The Invader™ assay uses a novel linear signal amplificationtechnology that improves upon the long turnaround times required of thetypical PCR DNA sequenced-based analysis. See Cooksey et al.,Antimicrobial Agents and Chemotherapy 44:1296-1301 (2000). This assay isbased on cleavage of a unique secondary structure formed between twooverlapping oligonucleotides that hybridize to the target sequence ofinterest to form a “flap.” Each “flap” then generates thousands ofsignals per hour. Thus, the results of this technique can be easilyread, and the methods do not require exponential amplification of theDNA target. The Invader™ system uses two short DNA probes, which arehybridized to a DNA target. The structure formed by the hybridizationevent is recognized by a special cleavase enzyme that cuts one of theprobes to release a short DNA “flap.” Each released “flap” then binds toa fluorescently-labeled probe to form another cleavage structure. Whenthe cleavase enzyme cuts the labeled probe, the probe emits a detectablefluorescence signal. See e.g. Lyamichev et al., Nat. Biotechnol.,17:292-296 (1999).

The rolling circle method is another method that avoids exponentialamplification. Lizardi et al., Nature Genetics, 19:225-232 (1998) (whichis incorporated herein by reference). For example, Sniper™, a commercialembodiment of this method, is a sensitive, high-throughput SNP scoringsystem designed for the accurate fluorescent detection of specificvariants. For each nucleotide variant, two linear, allele-specificprobes are designed. The two allele-specific probes are identical withthe exception of the 3′-base, which is varied to complement the variantsite. In the first stage of the assay, target DNA is denatured and thenhybridized with a pair of single, allele-specific, open-circleoligonucleotide probes. When the 3′-base exactly complements the targetDNA, ligation of the probe will preferentially occur. Subsequentdetection of the circularized oligonucleotide probes is by rollingcircle amplification, whereupon the amplified probe products aredetected by fluorescence. See Clark and Pickering, Life Science News 6,2000, Amersham Pharmacia Biotech (2000).

A number of other techniques that avoid amplification all togetherinclude, e.g., surface-enhanced resonance Raman scattering (SERRS),fluorescence correlation spectroscopy, and single-moleculeelectrophoresis. In SERRS, a chromophore-nucleic acid conjugate isabsorbed onto colloidal silver and is irradiated with laser light at aresonant frequency of the chromophore. See Graham et al., Anal. Chem.,69:4703-4707 (1997). The fluorescence correlation spectroscopy is basedon the spatio-temporal correlations among fluctuating light signals andtrapping single molecules in an electric field. See Eigen et al., Proc.Natl. Acad. Sci. USA, 91:5740-5747 (1994). In single-moleculeelectrophoresis, the electrophoretic velocity of a fluorescently taggednucleic acid is determined by measuring the time required for themolecule to travel a predetermined distance between two laser beams. SeeCastro et al., Anal. Chem., 67:3181-3186 (1995).

In addition, the allele-specific oligonucleotides (ASO) can also be usedin in situ hybridization using tissues or cells as samples. Theoligonucleotide probes which can hybridize differentially with thewild-type gene sequence or the gene sequence harboring a mutation may belabeled with radioactive isotopes, fluorescence, or other detectablemarkers. In situ hybridization techniques are well known in the art andtheir adaptation to the present invention for detecting the presence orabsence of a nucleotide variant in the one or more gene of a particularindividual should be apparent to a skilled artisan apprised of thisdisclosure.

Accordingly, the presence or absence of one or more genes nucleotidevariant or amino acid variant in an individual can be determined usingany of the detection methods described above.

Typically, once the presence or absence of one or more gene nucleotidevariants or amino acid variants is determined, physicians or geneticcounselors or patients or other researchers may be informed of theresult. Specifically the result can be cast in a transmittable form thatcan be communicated or transmitted to other researchers or physicians orgenetic counselors or patients. Such a form can vary and can be tangibleor intangible. The result with regard to the presence or absence of anucleotide variant of the present invention in the individual tested canbe embodied in descriptive statements, diagrams, photographs, charts,images or any other visual forms. For example, images of gelelectrophoresis of PCR products can be used in explaining the results.Diagrams showing where a variant occurs in an individual's gene are alsouseful in indicating the testing results. The statements and visualforms can be recorded on a tangible media such as papers, computerreadable media such as floppy disks, compact disks, etc., or on anintangible media, e.g., an electronic media in the form of email orwebsite on internet or intranet. In addition, the result with regard tothe presence or absence of a nucleotide variant or amino acid variant inthe individual tested can also be recorded in a sound form andtransmitted through any suitable media, e.g., analog or digital cablelines, fiber optic cables, etc., via telephone, facsimile, wirelessmobile phone, internet phone and the like.

Thus, the information and data on a test result can be produced anywherein the world and transmitted to a different location. For example, whena genotyping assay is conducted offshore, the information and data on atest result may be generated and cast in a transmittable form asdescribed above. The test result in a transmittable form thus can beimported into the U.S. Accordingly, the present invention alsoencompasses a method for producing a transmittable form of informationon the genotype of the two or more suspected cancer samples from anindividual. The method comprises the steps of (1) determining thegenotype of the DNA from the samples according to methods of the presentinvention; and (2) embodying the result of the determining step in atransmittable form. The transmittable form is the product of theproduction method.

In Situ Hybridization

In situ hybridization assays are well known and are generally describedin Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells, e.g., from a biopsy, are fixed to a solidsupport, typically a glass slide. If DNA is to be probed, the cells aredenatured with heat or alkali. The cells are then contacted with ahybridization solution at a moderate temperature to permit annealing ofspecific probes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters. FISH (fluorescence in situhybridization) uses fluorescent probes that bind to only those parts ofa sequence with which they show a high degree of sequence similarity.

In situ hybridization can be used to detect specific gene sequences intissue sections or cell preparations by hybridizing the complementarystrand of a nucleotide probe to the sequence of interest. Fluorescent insitu hybridization (FISH) uses a fluorescent probe to increase thesensitivity of in situ hybridization.

FISH is a cytogenetic technique used to detect and localize specificpolynucleotide sequences in cells. For example, FISH can be used todetect DNA sequences on chromosomes. FISH can also be used to detect andlocalize specific RNAs, e.g., mRNAs, within tissue samples. In FISH usesfluorescent probes that bind to specific nucleotide sequences to whichthey show a high degree of sequence similarity. Fluorescence microscopycan be used to find out whether and where the fluorescent probes arebound. In addition to detecting specific nucleotide sequences, e.g.,translocations, fusion, breaks, duplications and other chromosomalabnormalities, FISH can help define the spatial-temporal patterns ofspecific gene copy number and/or gene expression within cells andtissues.

Various types of FISH probes can be used to detect chromosometranslocations. Dual color, single fusion probes can be useful indetecting cells possessing a specific chromosomal translocation. The DNAprobe hybridization targets are located on one side of each of the twogenetic breakpoints. “Extra signal” probes can reduce the frequency ofnormal cells exhibiting an abnormal FISH pattern due to the randomco-localization of probe signals in a normal nucleus. One large probespans one breakpoint, while the other probe flanks the breakpoint on theother gene. Dual color, break apart probes are useful in cases wherethere may be multiple translocation partners associated with a knowngenetic breakpoint. This labeling scheme features two differentlycolored probes that hybridize to targets on opposite sides of abreakpoint in one gene. Dual color, dual fusion probes can reduce thenumber of normal nuclei exhibiting abnormal signal patterns. The probeoffers advantages in detecting low levels of nuclei possessing a simplebalanced translocation. Large probes span two breakpoints on differentchromosomes. Such probes are available as Vysis probes from AbbottLaboratories, Abbott Park, Ill.

Comparative Genomic Hybridization (CGH) comprises a molecularcytogenetic method of screening tumor samples for genetic changesshowing characteristic patterns for copy number changes at chromosomaland subchromosomal levels. Alterations in patterns can be classified asDNA gains and losses. CGH employs the kinetics of in situ hybridizationto compare the copy numbers of different DNA or RNA sequences from asample, or the copy numbers of different DNA or RNA sequences in onesample to the copy numbers of the substantially identical sequences inanother sample. In many useful applications of CGH, the DNA or RNA isisolated from a subject cell or cell population. The comparisons can bequalitative or quantitative. Procedures are described that permitdetermination of the absolute copy numbers of DNA sequences throughoutthe genome of a cell or cell population if the absolute copy number isknown or determined for one or several sequences. The differentsequences are discriminated from each other by the different locationsof their binding sites when hybridized to a reference genome, usuallymetaphase chromosomes but in certain cases interphase nuclei. The copynumber information originates from comparisons of the intensities of thehybridization signals among the different locations on the referencegenome. The methods, techniques and applications of CGH are known, suchas described in U.S. Pat. No. 6,335,167, and in U.S. App. Ser. No.60/804,818, the relevant parts of which are herein incorporated byreference.

In an embodiment, CGH used to compare nucleic acids between diseased andhealthy tissues. The method comprises isolating DNA from disease tissues(e.g., tumors) and reference tissues (e.g., healthy tissue) and labelingeach with a different “color” or fluor. The two samples are mixed andhybridized to normal metaphase chromosomes. In the case of array ormatrix CGH, the hybridization mixing is done on a slide with thousandsof DNA probes. A variety of detection system can be used that basicallydetermine the color ratio along the chromosomes to determine DNA regionsthat might be gained or lost in the diseased samples as compared to thereference.

Data and Analysis

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd ed., 2001). See U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention relates to embodiments that includemethods for providing genetic information over networks such as theInternet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (U.S.Publication Number 20020183936), 10/065,856, 10/065,868, 10/328,818,10/328,872, 10/423,403, and 60/482,389. For example, one or moremolecular profiling techniques can be performed in one location, e.g., acity, state, country or continent, and the results can be transmitted toa different city, state, country or continent. Treatment selection canthen be made in whole or in part in the second location. The methods ofthe invention comprise transmittal of information between differentlocations.

Molecular Profiling for Treatment Selection

The methods of the invention provide a candidate treatment selection fora subject in need thereof. Molecular profiling can be used to identifyone or more candidate therapeutic agents for an individual sufferingfrom a condition in which one or more of the biomarkers disclosed hereinare targets for treatment. For example, the method can identify one ormore chemotherapy treatments for a cancer. In an aspect, the inventionprovides a method comprising: performing an immunohistochemistry (IHC)analysis on a sample from the subject to determine an IHC expressionprofile on at least five proteins; performing a microarray analysis onthe sample to determine a microarray expression profile on at least tengenes; performing a fluorescent in-situ hybridization (FISH) analysis onthe sample to determine a FISH mutation profile on at least one gene;performing DNA sequencing on the sample to determine a sequencingmutation profile on at least one gene; and comparing the IHC expressionprofile, microarray expression profile, FISH mutation profile andsequencing mutation profile against a rules database, wherein the rulesdatabase comprises a mapping of treatments whose biological activity isknown against diseased cells that: i) overexpress or underexpress one ormore proteins included in the IHC expression profile; ii) overexpress orunderexpress one or more genes included in the microarray expressionprofile; iii) have zero or more mutations in one or more genes includedin the FISH mutation profile; and/or iv) have zero or more mutations inone or more genes included in the sequencing mutation profile; andidentifying the treatment if the comparison against the rules databaseindicates that the treatment should have biological activity against thediseased cells; and the comparison against the rules database does notcontraindicate the treatment for treating the diseased cells. Thedisease can be a cancer. The molecular profiling steps can be performedin any order. In some embodiments, not all of the molecular profilingsteps are performed. As a non-limiting example, microarray analysis isnot performed if the sample quality does not meet a threshold value, asdescribed herein. In another example, sequencing is performed only ifFISH analysis meets a threshold value. Any relevant biomarker can beassessed using one or more of the molecular profiling techniquesdescribed herein or known in the art. The marker need only have somedirect or indirect association with a treatment to be useful.

Molecular profiling comprises the profiling of at least one gene (orgene product) for each assay technique that is performed. Differentnumbers of genes can be assayed with different techniques. Any markerdisclosed herein that is associated directly or indirectly with a targettherapeutic can be assessed. For example, any “druggable target”comprising a target that can be modulated with a therapeutic agent suchas a small molecule or binding agent such as an antibody, is a candidatefor inclusion in the molecular profiling methods of the invention. Thetarget can also be indirectly drug associated, such as a component of abiological pathway that is affected by the associated drug. Themolecular profiling can be based on either the gene, e.g., DNA sequence,and/or gene product, e.g., mRNA or protein. Such nucleic acid and/orpolypeptide can be profiled as applicable as to presence or absence,level or amount, activity, mutation, sequence, haplotype, rearrangement,copy number, or other measurable characteristic. In some embodiments, asingle gene and/or one or more corresponding gene products is assayed bymore than one molecular profiling technique. A gene or gene product(also referred to herein as “marker” or “biomarker”), e.g., an mRNA orprotein, is assessed using applicable techniques (e.g., to assess DNA,RNA, protein), including without limitation FISH, microarray, IHC,sequencing or immunoassay. Therefore, any of the markers disclosedherein can be assayed by a single molecular profiling technique or bymultiple methods disclosed herein (e.g., a single marker is profiled byone or more of IHC, FISH, sequencing, microarray, etc.). In someembodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or at least about 100genes or gene products are profiled by at least one technique, aplurality of techniques, or using a combination of FISH, microarray,IHC, and sequencing. In some embodiments, at least about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000,17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000,26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000,35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000,44,000, 45,000, 46,000, 47,000, 48,000, 49,000, or at least 50,000 genesor gene products are profiled using various techniques. The number ofmarkers assayed can depend on the technique used. For example,microarray and massively parallel sequencing lend themselves to highthroughput analysis. Because molecular profiling queries molecularcharacteristics of the tumor itself, this approach provides informationon therapies that might not otherwise be considered based on the lineageof the tumor.

In some embodiments, a sample from a subject in need thereof is profiledusing methods which include but are not limited to IHC expressionprofiling, microarray expression profiling, FISH mutation profiling,and/or sequencing mutation profiling (such as by PCR, RT-PCR,pyrosequencing) for one or more of the following: ABCC1, ABCG2, ACE2,ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1, beta IIItubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33,CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK 17, CK5/6, c-KIT, c-Met, c-Myc, COX-2, Cyclin D1, DCK, DHFR, DNMT1, DNMT3A,DNMT3B, E-Cadherin, ECGF1, EGFR, EML4-ALK fusion, EPHA2, Epiregulin, ER,ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2,FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1,hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R,IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT,K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN, MET, MGMT, MLH1, MMR,MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, NRAS, ODC1, OGFR,p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR,PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, PTPN12, RAF1, RARA,RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3,SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS, TUBB3,TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, ZAP70.

Table 21 provides a listing of gene and corresponding protein symbolsand names of many of the molecular profiling targets that are analyzedaccording to the methods of the invention. As understood by those ofskill in the art, genes and proteins have developed a number ofalternative names in the scientific literature. Thus, the listing inTable 2 comprises an illustrative but not exhaustive compilation. Afurther listing of gene aliases and descriptions can be found using avariety of online databases, including GeneCards® (www.genecards.org),HUGO Gene Nomenclature (www.genenames.org), Entrez Gene(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), UniProtKB/Swiss-Prot(www.uniprot.org), UniProtKB/TrEMBL (www.uniprot.org), OMIM(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), GeneLoc(genecards.weizmann.ac.illgeneloc/), and Ensembl (www.ensembl.org).Generally, gene symbols and names below correspond to those approved byHUGO, and protein names are those recommended by UniProtKB/Swiss-Prot.Common alternatives are provided as well. Where a protein name indicatesa precursor, the mature protein is also implied. Throughout theapplication, gene and protein symbols may be used interchangeably andthe meaning can be derived from context, e.g., FISH is used to analyzenucleic acids whereas IHC is used to analyze protein.

TABLE 2 Gene and Protein Names Gene Protein Symbol Gene Name SymbolProtein Name ABCB1, ATP-binding cassette, sub-family B ABCB1, Multidrugresistance protein 1; P- PGP (MDR/TAP), member 1 MDR1, glycoprotein PGPABCC1, ATP-binding cassette, sub-family C MRP1, Multidrugresistance-associated protein MRP1 (CFTR/MRP), member 1 ABCC1 1 ABCG2,ATP-binding cassette, sub-family G ABCG2 ATP-binding cassette sub-familyG BCRP (WHITE), member 2 member 2 ACE2 angiotensin I converting enzymeACE2 Angiotensin-converting enzyme 2 (peptidyl-dipeptidase A) 2precursor ADA adenosine deaminase ADA Adenosine deaminase ADH1C alcoholdehydrogenase 1C (class I), ADH1G Alcohol dehydrogenase 1C gammapolypeptide ADH4 alcohol dehydrogenase 4 (class II), ADH4 Alcoholdehydrogenase 4 pi polypeptide AGT angiotensinogen (serpin peptidaseANGT, Angiotensinogen precursor inhibitor, clade A, member 8) AGT ALKanaplastic lymphoma receptor ALK ALK tyrosine kinase receptor tyrosinekinase precursor AR androgen receptor AR Androgen receptor AREGamphiregulin AREG Amphiregulin precursor ASNS asparagine synthetase ASNSAsparagine synthetase [glutamine- hydrolyzing] BCL2 B-cell CLL/lymphoma2 BCL2 Apoptosis regulator Bcl-2 BDCA1, CD1c molecule CD1C T-cellsurface glycoprotein CD1c CD1C precursor BIRC5 baculoviral IAPrepeat-containing 5 BIRC5, Baculoviral IAP repeat-containing Survivinprotein 5; Survivin BRAF v-raf murine sarcoma viral B-RAF,Serine/threonine-protein kinase B-raf oncogene homolog B1 BRAF BRCA1breast cancer 1, early onset BRCA1 Breast cancer type 1 susceptibilityprotein BRCA2 breast cancer 2, early onset BRCA2 Breast cancer type 2susceptibility protein CA2 carbonic anhydrase II CA2 Carbonic anhydrase2 CAV1 caveolin 1, caveolae protein, CAV1 Caveolin-1 22 kDa CCND1 cyclinD1 CCND1, G1/S-specific cyclin-D1 Cyclin D1, BCL-1 CD20,membrane-spanning 4-domains, CD20 B-lymphocyte antigen CD20 MS4A1subfamily A, member 1 CD25, interleukin 2 receptor, alpha CD25Interleukin-2 receptor subunit alpha IL2RA precursor CD33 CD33 moleculeCD33 Myeloid cell surface antigen CD33 precursor CD52, CD52 moleculeCD52 CAMPATH-1 antigen precursor CDW52 CDA cytidine deaminase CDACytidine deaminase CDH1, cadherin 1, type 1, E-cadherin E-Cad Cadherin-1precursor (E-cadherin) ECAD (epithelial) CDK2 cyclin-dependent kinase 2CDK2 Cell division protein kinase 2 CDKN1A, cyclin-dependent kinaseinhibitor CDKN1A, Cyclin-dependent kinase inhibitor 1 P21 1A (p21, Cip1)p21 CDKN1B cyclin-dependent kinase inhibitor CDKN1B, Cyclin-dependentkinase inhibitor 1B 1B (p27, Kip1) p27 CDKN2A, cyclin-dependent kinaseinhibitor CD21A, Cyclin-dependent kinase inhibitor 2A, P16 2A (melanoma,p16, inhibits p16 isoforms 1/2/3 CDK4) CES2 carboxylesterase 2(intestine, liver) CES2, Carboxylesterase 2 precursor EST2 CK 5/6cytokeratin 5/cytokeratin 6 CK 5/6 Keratin, type II cytoskeletal 5;Keratin, type II cytoskeletal 6 CK14, keratin 14 CK14 Keratin, type Icytoskeletal 14 KRT14 CK17, keratin 17 CK17 Keratin, type I cytoskeletal17 KRT17 COX2, prostaglandin-endoperoxide COX-2, Prostaglandin G/Hsynthase 2 PTGS2 synthase 2 (prostaglandin G/H PTGS2 precursor synthaseand cyclooxygenase) DCK deoxycytidine kinase DCK Deoxycytidine kinaseDHFR dihydrofolate reductase DHFR Dihydrofolate reductase DNMT1 DNA(cytosine-5-)- DNMT1 DNA (cytosine-5)-methyltransferase 1methyltransferase 1 DNMT3A DNA (cytosine-5-)- DNMT3A DNA(cytosine-5)-methyltransferase 3A methyltransferase 3 alpha DNMT3B DNA(cytosine-5-)- DNMT3B DNA (cytosine-5)-methyltransferasemethyltransferase 3 beta 3B ECGF1, thymidine phosphorylase TYMP,Thymidine phosphorylase precursor TYMP PD-ECGF, ECDF1 EGFR, epidermalgrowth factor receptor EGFR, Epidermal growth factor receptor ERBB1,(erythroblastic leukemia viral (v- ERBB1, precursor HER1 erb-b) oncogenehomolog, avian) HER1 EML4 echinoderm microtubule associated EML4Echinoderm microtubule-associated protein like 4 protein-like 4 EPHA2EPH receptor A2 EPHA2 Ephrin type-A receptor 2 precursor ER, estrogenreceptor 1 ER, Estrogen receptor ESR1 ESR1 ERBB2, v-erb-b2erythroblastic leukemia ERBB2, Receptor tyrosine-protein kinase erbB-HER2/ viral oncogene homolog 2, HER2, 2 precursor NEU neuro/glioblastomaderived HER-2/ oncogene homolog (avian) neu ERCC1 excision repair cross-ERCC1 DNA excision repair protein ERCC-1 complementing rodent repairdeficiency, complementation group 1 (includes overlapping antisensesequence) ERCC3 excision repair cross- ERCC3 TFIIH basal transcriptionfactor complementing rodent repair complex helicase XPB subunitdeficiency, complementation group 3 (xeroderma pigmentosum group Bcomplementing) EREG Epiregulin EREG Proepiregulin precursor FLT1fins-related tyrosine kinase 1 FLT-1, Vascular endothelial growth factor(vascular endothelial growth VEGFR1 receptor 1 precursor factor/vascularpermeability factor receptor) FOLR1 folate receptor 1 (adult) FOLR1Folate receptor alpha precursor FOLR2 folate receptor 2 (fetal) FOLR2Folate receptor beta precursor FSHB follicle stimulating hormone, betaFSHB Follitropin subunit beta precursor polypeptide FSHPRH1, centromereprotein I FSHPRH1, Centromere protein I CENP1 CENP1 FSHR folliclestimulating hormone FSHR Follicle-stimulating hormone receptor receptorprecursor FYN FYN oncogene related to SRC, FYN Tyrosine-protein kinaseFyn FGR, YES GART phosphoribosylglycinamide GART, Trifunctional purinebiosynthetic formyltransferase, PUR2 protein adenosine-3phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazolesynthetase GNRH1 gonadotropin-releasing hormone 1 GNRH1,Progonadoliberin-1 precursor (luteinizing-releasing hormone) GON1GNRHR1, gonadotropin-releasing hormone GNRHR1 Gonadotropin-releasinghormone GNRHR receptor receptor GSTP1 glutathione S-transferase pi 1GSTP1 Glutathione S-transferase P HCK hemopoietic cell kinase HCKTyrosine-protein kinase HCK HDAC1 histone deacetylase 1 HDAC1 Histonedeacetylase 1 HGF hepatocyte growth factor HGF Hepatocyte growth factorprecursor (hepapoietin A; scatter factor) HIF1A hypoxia inducible factor1, alpha HIF1A Hypoxia-inducible factor 1-alpha subunit (basichelix-loop-helix transcription factor) HIG1, HIG1 hypoxia inducibledomain HIG1, HIG1 domain family member 1A HIGD1A, family, member 1AHIGD1A, HIG1A HIG1A HSP90AA1, heat shock protein 90 kDa alpha HSP90,Heat shock protein HSP 90-alpha HSP90, (cytosolic), class A member 1HSP90A HSPCA IGF1R insulin-like growth factor 1 receptor IGF-1RInsulin-like growth factor 1 receptor precursor IGFBP3, insulin-likegrowth factor binding IGFBP-3, Insulin-like growth factor-bindingIGFRBP3 protein 3 IBP-3 protein 3 precursor IGFBP4, insulin-like growthfactor binding IGFBP-4, Insulin-like growth factor-binding IGFRBP4protein 4 IBP-4 protein 4 precursor IGFBP5, insulin-like growth factorbinding IGFBP-5, Insulin-like growth factor-binding IGFRBP5 protein 5IBP-5 protein 5 precursor IL13RA1 interleukin 13 receptor, alpha 1IL-13RA1 Interleukin-13 receptor subunit alpha-1 precursor KDR kinaseinsert domain receptor (a KDR, Vascular endothelial growth factor typeIII receptor tyrosine kinase) VEGFR2 receptor 2 precursor KIT, v-kitHardy-Zuckerman 4 feline KIT, Mast/stem cell growth factor receptorc-KIT sarcoma viral oncogene homolog c-KIT, precursor CD117, SCFR KRASv-Ki-ras2 Kirsten rat sarcoma viral K-RAS GTPase KRas precursor oncogenehomolog LCK lymphocyte-specific protein LCK Tyrosine-protein kinase Lcktyrosine kinase LTB lymphotoxin beta (TNF LTB, Lymphotoxin-betasuperfamily, member 3) TNF3 LTBR lymphotoxin beta receptor (TNFR LTBR,Tumor necrosis factor receptor superfamily, member 3) LTBR3, superfamilymember 3 precursor TNFR LYN v-yes-1 Yamaguchi sarcoma viral LYNTyrosine-protein kinase Lyn related oncogene homolog MET, metproto-oncogene (hepatocyte MET, Hepatocyte growth factor receptor c-METgrowth factor receptor) c-MET precursor MGMT O-6-methylguanine-DNA MGMTMethylated-DNA--protein-cysteine methyltransferase methyltransferaseMKI67, antigen identified by monoclonal Ki67, Antigen KI-67 KI67antibody Ki-67 Ki-67 MLH1 mutL homolog 1, colon cancer, MLH1 DNAmismatch repair protein Mlh1 nonpolyposis type 2 (E. coli) MMR mismatchrepair (refers to MLH1, MSH2, MSH5) MSH2 mutS homolog 2, colon cancer,MSH2 DNA mismatch repair protein Msh2 nonpolyposis type 1 (E. coli) MSH5mutS homolog 5 (E. coli) MSH5, MutS protein homolog 5 hMSH5 MYC, v-mycmyelocytomatosis viral MYC, Myc proto-oncogene protein c-MYC oncogenehomolog (avian) c-MYC NBN, P95 nibrin NBN, p95 Nibrin NDGR1 N-mycdownstream regulated 1 NDGR1 Protein NDGR1 NFKB1 nuclear factor of kappalight NFKB1 Nuclear factor NF-kappa-B p105 polypeptide gene enhancer inB- subunit cells 1 NFKB2 nuclear factor of kappa light NFKB2 Nuclearfactor NF-kappa-B p100 polypeptide gene enhancer in B- subunit cells 2(p49/p100) NFKBIA nuclear factor of kappa light NFKBIA NF-kappa-Binhibitor alpha polypeptide gene enhancer in B- cells inhibitor, alphaNRAS neuroblastoma RAS viral (v-ras) NRAS GTPase NRas, Transformingprotein oncogene homolog N-Ras ODC1 ornithine decarboxylase 1 ODCOrnithine decarboxylase OGFR opioid growth factor receptor OGFR Opioidgrowth factor receptor PARP1 poly (ADP-ribose) polymerase 1 PARP-1 Poly[ADP-ribose] polymerase 1 PDGFC platelet derived growth factor C PDGF-C,Platelet-derived growth factor C VEGF-E precursor PDGFR platelet-derivedgrowth factor PDGFR Platelet-derived growth factor receptor receptorPDGFRA platelet-derived growth factor PDGFRA, Alpha-typeplatelet-derived growth receptor, alpha polypeptide PDGFR2, factorreceptor precursor CD140 A PDGFRB platelet-derived growth factor PDGFRB,Beta-type platelet-derived growth receptor, beta polypeptide PDGFR,factor receptor precursor PDGFR1, CD140 B PGR progesterone receptor PRProgesterone receptor PIK3CA phosphoinositide-3-kinase, PI3KPhosphoinositide-3-kinase, catalytic, catalytic, alpha polypeptidesubunit alpha polypeptide p110α POLA1 polymerase (DNA directed), alphaPOLA, DNA polymerase alpha catalytic 1, catalytic subunit; polymerasePOLA1, subunit (DNA directed), alpha, polymerase p180 (DNA directed),alpha 1 PPARG, peroxisome proliferator-activated PPARG Peroxisomeproliferator-activated PPARG1, receptor gamma receptor gamma PPARG2,PPAR- gamma, NR1C3 PPARGC1A, peroxisome proliferator-activated PGC-1-Peroxisome proliferator-activated LEM6, receptor gamma, coactivator 1alpha, receptor gamma coactivator 1-alpha; PGC1, alpha PPARGC-PPAR-gamma coactivator 1-alpha PGC1A, 1-alpha PPARGC1 PSMD9, proteasome(prosome, macropain) p27 26S proteasome non-ATPase P27 26S subunit,non-ATPase, 9 regulatory subunit 9 PTEN, phosphatase and tensin homologPTEN Phosphatidylinositol-3,4,5- MMAC1, trisphosphate 3-phosphatase anddual- TEP1 specificity protein phosphatase; Mutated in multiple advancedcancers 1 PTPN12 protein tyrosine phosphatase, non- PTPG1Tyrosine-protein phosphatase non- receptor type 12 receptor type 12;Protein-tyrosine phosphatase G1 RAF1 v-raf-1 murine leukemia viral RAF,RAF proto-oncogene serine/threonine- oncogene homolog 1 RAF-1, proteinkinase c-RAF RARA retinoic acid receptor, alpha RAR, Retinoic acidreceptor alpha RAR- alpha, RARA RRM1 ribonucleotide reductase M1 RRM1,Ribonucleoside-diphosphate reductase RR1 large subunit RRM2ribonucleotide reductase M2 RRM2, Ribonucleoside-diphosphate reductaseRR2M, subunit M2 RR2 RRM2B ribonucleotide reductase M2 B RRM2B,Ribonucleoside-diphosphate reductase (TP53 inducible) P53R2 subunit M2 BRXRB retinoid X receptor, beta RXRB Retinoic acid receptor RXR-beta RXRGretinoid X receptor, gamma RXRG, Retinoic acid receptor RXR-gamma RXRCSIK2 salt-inducible kinase 2 SIK2, Salt-inducible protein kinase 2;Q9H0K1 Serine/threonine-protein kinase SIK2 SLC29A1 solute carrierfamily 29 (nucleoside ENT-1 Equilibrative nucleoside transporter 1transporters), member 1 SPARC secreted protein, acidic, cysteine- SPARCSPARC precursor; Osteonectin rich (osteonectin) SRC v-src sarcoma(Schmidt-Ruppin A- SRC Proto-oncogene tyrosine-protein kinase 2) viraloncogene homolog (avian) Src SSTR1 somatostatin receptor 1 SSTR1,Somatostatin receptor type 1 SSR1, SS1R SSTR2 somatostatin receptor 2SSTR2, Somatostatin receptor type 2 SSR2, SS2R SSTR3 somatostatinreceptor 3 SSTR3, Somatostatin receptor type 3 SSR3, SS3R SSTR4somatostatin receptor 4 SSTR4, Somatostatin receptor type 4 SSR4, SS4RSSTR5 somatostatin receptor 5 SSTR5, Somatostatin receptor type 5 SSR5,SS5R TK1 thymidine kinase 1, soluble TK1, Thymidine kinase, cytosolicKITH TLE3 transducin-like enhancer of split 3 TLE3 Transducin-likeenhancer protein 3 (E(sp1) homolog, Drosophila) TNF tumor necrosisfactor (TNF TNF, Tumor necrosis factor precursor superfamily, member 2)TNF-alpha, TNF-a TOP1, topoisomerase (DNA) I TOP1, DNA topoisomerase 1TOPO1 TOPO1 TOP2A, topoisomerase (DNA) II alpha TOP2A, DNA topoisomerase2-alpha; TOPO2A 170 kDa TOP2, Topoisomerase II alpha TOPO2A TOP2B,topoisomerase (DNA) II beta TOP2B, DNA topoisomerase 2-beta; TOPO2B 180kDa TOPO2B Topoisomerase II beta TP53 tumor protein p53 p53 Cellulartumor antigen p53 TUBB3 tubulin, beta 3 Beta III Tubulin beta-3 chaintubulin, TUBB3, TUBB4 TXN thioredoxin TXN, Thioredoxin TRX, TRX-1 TXNRD1thioredoxin reductase 1 TXNRD1, Thioredoxin reductase 1, cytoplasmic;TXNR Oxidoreductase TYMS, thymidylate synthetase TYMS, Thymidylatesynthase TS TS VDR vitamin D (1,25-dihydroxyvitamin VDR Vitamin D3receptor D3) receptor VEGFA, vascular endothelial growth factor VEGF-A,Vascular endothelial growth factor A VEGF A VEGF precursor VEGFCvascular endothelial growth factor VEGF-C Vascular endothelial growthfactor C C precursor VHL von Hippel-Lindau tumor VHL Von Hippel-Lindaudisease tumor suppressor suppressor YES1 v-yes-1 Yamaguchi sarcoma viralYES 1, Proto-oncogene tyrosine-protein kinase oncogene homolog 1 Yes,Yes p61-Yes ZAP70 zeta-chain (TCR) associated protein ZAP-70Tyrosine-protein kinase ZAP-70 kinase 70 kDa

In some embodiments, additional molecular profiling methods areperformed. These can include without limitation PCR, RT-PCR, Q-PCR,SAGE, MPSS, immunoassays and other techniques to assess biologicalsystems described herein or known to those of skill in the art. Thechoice of genes and gene products to be assayed can be updated over timeas new treatments and new drug targets are identified. Once theexpression or mutation of a biomarker is correlated with a treatmentoption, it can be assessed by molecular profiling. One of skill willappreciate that such molecular profiling is not limited to thosetechniques disclosed herein but comprises any methodology conventionalfor assessing nucleic acid or protein levels, sequence information, orboth. The methods of the invention can also take advantage of anyimprovements to current methods or new molecular profiling techniquesdeveloped in the future. In some embodiments, a gene or gene product isassessed by a single molecular profiling technique. In otherembodiments, a gene and/or gene product is assessed by multiplemolecular profiling techniques. In a non-limiting example, a genesequence can be assayed by one or more of FISH and pyrosequencinganalysis, the mRNA gene product can be assayed by one or more of RT-PCRand microarray, and the protein gene product can be assayed by one ormore of IHC and immunoassay. One of skill will appreciate that anycombination of biomarkers and molecular profiling techniques that willbenefit disease treatment are contemplated by the invention.

Genes and gene products that are known to play a role in cancer and canbe assayed by any of the molecular profiling techniques of the inventioninclude without limitation 2AR, A DISINTEGRIN, ACTIVATOR OF THYROID ANDRETINOIC ACID RECEPTOR (ACTR), ADAM 11, ADIPOGENESIS INHIBITORY FACTOR(ADIF), ALPHA 6 INTEGRIN SUBUNIT, ALPHA V INTEGRIN SUBUNIT,ALPHA-CATENIN, AMPLIFIED IN BREAST CANCER 1 (AIB1), AMPLIFIED IN BREASTCANCER 3 (AIB3), AMPLIFIED IN BREAST CANCER 4 (AIB4), AMYLOID PRECURSORPROTEIN SECRETASE (APPS), AP-2 GAMMA, APPS, ATP-BINDING CASSETTETRANSPORTER (ABCT), PLACENTA-SPECIFIC (ABCP), ATP-BINDING CASSETTESUBFAMILY C MEMBER (ABCC1), BAG-1, BASIGIN (BSG), BCEI, B-CELLDIFFERENTIATION FACTOR (BCDF), B-CELL LEUKEMIA 2 (BCL-2), B-CELLSTIMULATORY FACTOR-2 (BSF-2), BCL-1, BCL-2-ASSOCIATED X PROTEIN (BAX),BCRP, BETA 1 INTEGRIN SUBUNIT, BETA 3 INTEGRIN SUBUNIT, BETA 5 INTEGRINSUBUNIT, BETA-2 INTERFERON, BETA-CATENIN, BETA-CATENIN, BONESIALOPROTEIN (BSP), BREAST CANCER ESTROGEN-INDUCIBLE SEQUENCE (BCEI),BREAST CANCER RESISTANCE PROTEIN (BCRP), BREAST CANCER TYPE 1 (BRCA1),BREAST CANCER TYPE 2 (BRCA2), BREAST CARCINOMA AMPLIFIED SEQUENCE 2(BCAS2), CADHERIN, EPITHELIAL CADHERIN-11, CADHERIN-ASSOCIATED PROTEIN,CALCITONIN RECEPTOR(CTR), CALCIUM PLACENTAL PROTEIN(CAPL), CALCYCLIN,CALLA, CAMS, CAPL, CARCINOEMBRYONIC ANTIGEN (CEA), CATENIN, ALPHA 1,CATHEPSIN B, CATHEPSIN D, CATHEPSIN K, CATHEPSIN L2, CATHEPSIN O,CATHEPSIN 01, CATHEPSIN V, CD10, CD146, CD147, CD24, CD29, CD44, CD51,CD54, CD61, CD66e, CD82, CD87, CD9, CEA, CELLULAR RETINOL-BINDINGPROTEIN 1 (CRBP1), c-ERBB-2, CK7, CK8, CK18, CK19, CK20, CLAUDIN-7,c-MET, COLLAGENASE, FIBROBLAST, COLLAGENASE, INTERSTITIAL,COLLAGENASE-3, COMMON ACUTE LYMPHOCYTIC LEUKEMIA ANTIGEN(CALLA),CONNEXIN 26 (Cx26), CONNEXIN 43 (Cx43), CORTACTIN, COX-2, CTLA-8, CTR,CTSD, CYCLIN D1, CYCLOOXYGENASE-2, CYTOKERATIN 18, CYTOKERATIN 19,CYTOKERATIN 8, CYTOTOXIC T-LYMPHOCYTE-ASSOCIATED SERINE ESTERASE 8(CTLA-8), DIFFERENTIATION—INHIBITING ACTIVITY (DIA), DNA AMPLIFIED INMAMMARY CARCINOMA 1 (DAM1), DNA TOPOISOMERASE II ALPHA, DR-NM23,E-CADHERIN, EMMPRIN, EMS1, ENDOTHELIAL CELL GROWTH FACTOR (ECGR),PLATELET-DERIVED (PD-ECGF), ENKEPHALINASE, EPIDERMAL GROWTH FACTORRECEPTOR (EGFR), EPISIALIN, EPITHELIAL MEMBRANE ANTIGEN (EMA), ER-ALPHA,ERBB2, ERBB4, ER-BETA, ERF-1, ERYTHROID-POTENTIATING ACTIVITY (EPA),ESR1, ESTROGEN RECEPTOR-ALPHA, ESTROGEN RECEPTOR-BETA, ETS-1,EXTRACELLULAR MATRIX METALLOPROTEINASE INDUCER (EMMPRIN), FIBRONECTINRECEPTOR, BETA POLYPEPTIDE (FNRB), FIBRONECTIN RECEPTOR BETA SUBUNIT(FNRB), FLK-1, GA15.3, GA733.2, GALECTIN-3, GAMMA-CATENIN, GAP JUNCTIONPROTEIN (26 kDa), GAP JUNCTION PROTEIN (43 kDa), GAP JUNCTION PROTEINALPHA-1 (GJA1), GAP JUNCTION PROTEIN BETA-2 (GJB2), GCP1, GELATINASE A,GELATINASE B, GELATINASE (72 kDa), GELATINASE (92 kDa), GLIOSTATIN,GLUCOCORTICOID RECEPTOR INTERACTING PROTEIN 1 (GRIP1), GLUTATHIONE5-TRANSFERASE p, GM-CSF, GRANULOCYTE CHEMOTACTIC PROTEIN 1 (GCP1),GRANULOCYTE-MACROPHAGE-COLONY STIMULATING FACTOR, GROWTH FACTOR RECEPTORBOUND-7 (GRB-7), GSTp, HAP, HEAT-SHOCK COGNATE PROTEIN 70 (HSC70),HEAT-STABLE ANTIGEN, HEPATOCYTE GROWTH FACTOR (HGF), HEPATOCYTE GROWTHFACTOR RECEPTOR (HGFR), HEPATOCYTE-STIMULATING FACTOR 111 (HSF III),HER-2, HER2/NEU, HERMES ANTIGEN, HET, HHM, HUMORAL HYPERCALCEMIA OFMALIGNANCY (HHM), ICERE-1, INT-1, INTERCELLULAR ADHESION MOLECULE-1(ICAM-1), INTERFERON-GAMMA-INDUCING FACTOR (IGIF), INTERLEUKIN-1 ALPHA(IL-1A), INTERLEUKIN-1 BETA (IL-1B), INTERLEUKIN-11 (IL-11),INTERLEUKIN-17 (IL-17), INTERLEUKIN-18 (IL-18), INTERLEUKIN-6 (IL-6),INTERLEUKIN-8 (IL-8), INVERSELY CORRELATED WITH ESTROGEN RECEPTOREXPRESSION-1 (ICERE-1), KAI1, KDR, KERATIN 8, KERATIN 18, KERATIN 19,KISS-1, LEUKEMIA INHIBITORY FACTOR (LIF), LIF, LOST IN INFLAMMATORYBREAST CANCER (LIBC), LOT (“LOST ON TRANSFORMATION”), LYMPHOCYTE HOMINGRECEPTOR, MACROPHAGE-COLONY STIMULATING FACTOR, MAGE-3, MAMMAGLOBIN,MASPIN, MC56, M-CSF, MDC, MDNCF, MDR, MELANOMA CELL ADHESION MOLECULE(MCAM), MEMBRANE METALLOENDOPEPTIDASE (MME), MEMBRANE-ASSOCIATED NEUTRALENDOPEPTIDASE (NEP), CYSTEINE-RICH PROTEIN (MDC), METASTASIN (MTS-1),MLN64, MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP13, MMP14, MMP15, MMP16,MMP17, MOESIN, MONOCYTE ARGININE-SERPIN, MONOCYTE-DERIVED NEUTROPHILCHEMOTACTIC FACTOR, MONOCYTE-DERIVED PLASMINOGEN ACTIVATOR INHIBITOR,MTS-1, MUC-1, MUC18, MUCIN LIKE CANCER ASSOCIATED ANTIGEN (MCA), MUCIN,MUC-1, MULTIDRUG RESISTANCE PROTEIN 1 (MDR, MDR1), MULTIDRUG RESISTANCERELATED PROTEIN-1 (MRP, MRP-1), N-CADHERIN, NEP, NEU, NEUTRALENDOPEPTIDASE, NEUTROPHIL-ACTIVATING PEPTIDE 1 (NAP1), NM23-H1, NM23-H2,NME1, NME2, NUCLEAR RECEPTOR COACTIVATOR-1 (NCoA-1), NUCLEAR RECEPTORCOACTIVATOR-2 (NCoA-2), NUCLEAR RECEPTOR COACTIVATOR-3 (NCoA-3),NUCLEOSIDE DIPHOSPHATE KINASE A (NDPKA), NUCLEOSIDE DIPHOSPHATE KINASE B(NDPKB), ONCOSTATIN M (OSM), ORNITHINE DECARBOXYLASE (ODC), OSTEOCLASTDIFFERENTIATION FACTOR (ODF), OSTEOCLAST DIFFERENTIATION FACTOR RECEPTOR(ODFR), OSTEONECTIN (OSN, ON), OSTEOPONTIN (OPN), OXYTOCIN RECEPTOR(OXTR), p27/kip1, p300/CBP COINTEGRATOR ASSOCIATE PROTEIN (p/CIP), p53,p9Ka, PAI-1, PAI-2, PARATHYROID ADENOMATOSIS1 (PRAD1), PARATHYROIDHORMONE-LIKE HORMONE (PTHLH), PARATHYROID HORMONE-RELATED PEPTIDE(PTHrP), P-CADHERIN, PD-ECGF, PDGF, PEANUT-REACTIVE URINARY MUCIN (PUM),P-GLYCOPROTEIN (P-GP), PGP-1, PHGS-2, PHS-2, PIP, PLAKOGLOBIN,PLASMINOGEN ACTIVATOR INHIBITOR (TYPE 1), PLASMINOGEN ACTIVATORINHIBITOR (TYPE 2), PLASMINOGEN ACTIVATOR (TISSUE-TYPE), PLASMINOGENACTIVATOR (UROKINASE-TYPE), PLATELET GLYCOPROTEIN IIIa (GP3A), PLAU,PLEOMORPHIC ADENOMA GENE-LIKE 1 (PLAGL1), POLYMORPHIC EPITHELIAL MUCIN(PEM), PRAD1, PROGESTERONE RECEPTOR (PgR), PROGESTERONE RESISTANCE,PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2, PROSTAGLANDIN G/H SYNTHASE-2,PROSTAGLANDIN H SYNTHASE-2, pS2, PS6K, PSORIASIN, PTHLH, PTHrP, RAD51,RAD52, RAD54, RAP46, RECEPTOR-ASSOCIATED COACTIVATOR3 (RAC3), REPRESSOROF ESTROGEN RECEPTOR ACTIVITY (REA), S100A4, S100A6, S100A7, S6K,SART-1, SCAFFOLD ATTACHMENT FACTOR B (SAF-B), SCATTER FACTOR(SF),SECRETED PHOSPHOPROTEIN-1 (SPP-1), SECRETED PROTEIN, ACIDIC AND RICH INCYSTEINE (SPARC), STANNICALCIN, STEROID RECEPTOR COACTIVATOR-1 (SRC-1),STEROID RECEPTOR COACTIVATOR-2 (SRC-2), STEROID RECEPTOR COACTIVATOR-3(SRC-3), STEROID RECEPTOR RNA ACTIVATOR(SRA), STROMELYSIN-1,STROMELYSIN-3, TENASCIN-C (TN-C), TESTES-SPECIFIC PROTEASE 50,THROMBOSPONDIN I, THROMBOSPONDIN II, THYMIDINE PHOSPHORYLASE (TP),THYROID HORMONE RECEPTOR ACTIVATOR MOLECULE 1 (TRAM-1), TIGHT JUNCTIONPROTEIN 1 (TJP1), TIMP1, TIMP2, TIMP3, TIMP4, TISSUE-TYPE PLASMINOGENACTIVATOR, TN-C, TP53, tPA, TRANSCRIPTIONAL INTERMEDIARY FACTOR2 (TIF2),TREFOIL FACTOR1 (TFF1), TSG101, TSP-1, TSP1, TSP-2, TSP2, TSP50, TUMORCELL COLLAGENASE STIMULATING FACTOR (TCSF), TUMOR-ASSOCIATED EPITHELIALMUCIN, uPA, uPAR, UROKINASE, UROKINASE-TYPE PLASMINOGEN ACTIVATOR,UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (uPAR), UVOMORULIN,VASCULAR ENDOTHELIAL GROWTH FACTOR, VASCULAR ENDOTHELIAL GROWTH FACTORRECEPTOR-2 (VEGFR2), VASCULAR ENDOTHELIAL GROWTH FACTOR-A, VASCULARPERMEABILITY FACTOR, VEGFR2, VERYLATE T-CELL ANTIGEN BETA (VLA-BETA),VIMENTIN, VITRONECTIN RECEPTOR ALPHA POLYPEPTIDE (VNRA), VITRONECTINRECEPTOR, VON WILLEBRAND FACTOR, VPF, VWF, WNT-1, ZAC, ZO-1, and ZONULAOCCLUDENS-1.

The gene products used for IHC expression profiling include withoutlimitation one or more of AR, BCRP, BCRP1, BRCA1, CAV-1, CK 5/6, CK14,CK17, c-Kit, cMET, cMYC, COX2, Cyclin D1, ECAD, EGFR, ER, ERCC1,Her2/Neu, IGF1R, IGFRBP1, IGFRBP2, IGFRBP3, IGFRBP4, IGFRBP5, IGFRBP6,IGFRBP7, Ki67, MGMT, MRP1, P53, P95, PDGFR, PDGFRA, PGP (MDR1), PR,PTEN, RRM1, SPARC, TLE3, TOP1, TOP2, TOP2A, TS, and TUBB3. In anembodiment, the IHC is performed on AR, BCRP, CAV-1, CK 5/6, CK14, CK17,c-Kit, COX2, Cyclin D1, ECAD, EGFR, ER, ERCC1, Her2/Neu, IGF1R, Ki67,MGMT, MRP1, P53, P95, PDGFRa, PGP (MDR1), PR, PTEN, RRM1, SPARC, TLE3,TOP1, TOP2A, TS, and TUBB3. In some embodiments, IHC analysis includesone or more of c-Met, EML4-ALK fusion, hENT-1, IGF-1R, MMR, p16, p21,p27, PARP-1, PI3K, and TLE3. IHC profiling of EGFR can also beperformed. IHC is also used to detect or test for various gene products,including without limitation one or more of the following: EGFR, SPARC,C-kit, ER, PR, Androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT,PDGFR, DCK, ERCC1, Thymidylate synthase, Her2/neu, or TOPO2A. In someembodiments, IHC is used to detect on or more of the following proteins,including without limitation: ADA, AR, ASNA, BCL2, BRCA2, c-Met, CD33,CDW52, CES2, DNMT1, EGFR, EML4-ALK fusion, ERBB2, ERCC3, ESR1, FOLR2,GART, GSTP1, HDAC1, hENT-1, HIF1A, HSPCA, IGF-1R, IL2RA, KIT, MLH1, MMR,MS4A1, MASH2, NFKB2, NFKBIA, OGFR, p16, p21, p27, PARP-1, PI3K, PDGFC,PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1,TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, orZAP70. The proteins can be detected by IHC using monoclonal orpolyclonal antibodies. In some embodiments, both are used. As anillustrative example, SPARC can be detected by anti-SPARC monoclonal(SPARC mono, SPARC m) and/or anti-SPARC polyclonal (SPARC poly, SPARC p)antibodies.

In some embodiments, IHC analysis according to the methods of theinvention includes one or more of AR, c-Kit, COX2, CAV-1, CK 5/6, CK14,CK17, ECAD, ER, Her2/Neu, Ki67, MRP1, P53, PDGFR, PGP, PR, PTEN, SPARC,TLE3 and TS. All of these genes can be examined. As indicated by initialresults of IHC or other molecular profiling methods as described herein,additional IHC assayscan be performed. In one embodiment, the additionalIHC comprises that of p95, or p95, Cyclin D1 and EGFR. IHC can also beperformed on IGFRBP3, IGFRBP4, IGFRBP5, or other forms of IGFRBP (e.g.,IGFRBP1, IGFRBP2, IGFRBP6, IGFRBP7). In another embodiment, theadditional IHC comprises that of one or more of BCRP, ERCC1, MGMT, P95,RRM1, TOP2A, and TOP1. In still another embodiment, the additional IHCcomprises that of one or more of BCRP, Cyclin D1, EGFR, ERCC1, MGMT,P95, RRM1, TOP2A, and TOP1. Any useful subset or all of these genes canbe examined. The additional IHC can be selected on the basis ofmolecular characteristics of the tumor so that IHC is only performedwhere it is likely to indicate a candidate therapy for treating thecancer. As described herein, the molecular characteristics of the tumordetermined can be determined by IHC combined with one or more of FISH,DNA microarray and mutation analysis. The genes and/or gene productsused for IHC analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genes and/or geneproducts listed in Table 2. The cancer can be an ovarian cancer. Thecancer can be a CUPS.

Microarray expression profiling can be used to simultaneously measurethe expression of one or more genes or gene products, including withoutlimitation ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33,CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2,ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK,HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK,LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1,PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1,RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4,SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL,YES1, and ZAP70. In some embodiments, the genes used for the microarrayexpression profiling comprise one or more of: EGFR, SPARC, C-kit, ER,PR, Androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK,ERCC1, Thymidylate synthase, Her2/neu, TOPO2A, ADA, AR, ASNA, BCL2,BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1, FOLR2, GART,GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2,NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA,RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR,VEGF, VHL, and ZAP70. One or more of the following genes can also beassessed by microarray expression profiling: ALK, EML4, hENT-1, IGF-1R,HSP90AA1, MMR, p16, p21, p27, PARP-1, PI3K and TLE3. The microarrayexpression profiling can be performed using a low density microarray, anexpression microarray, a comparative genomic hybridization (CGH)microarray, a single nucleotide polymorphism (SNP) microarray, aproteomic array an antibody array, or other array as disclosed herein orknown to those of skill in the art. In some embodiments, high throughputexpression arrays are used. Such systems include without limitationcommercially available systems from Agilent or Illumina, as described inmore detail herein.

Microarray expression profiling can be used to simultaneously measurethe expression of one or more genes or gene products, including withoutlimitation ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33,CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2,ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK,HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK,LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1,PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA,RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3,SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA,VHL, YES1, and ZAP70. The genes and/or gene products used for microarrayexpression profiling analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genesand/or gene products listed in Table 2. The cancer can be an ovariancancer. The cancer can be a CUPS.

FISH mutation profiling can be used to profile one or more of HER2,CMET, PIK3CA, EGFR, TOP2A, CMYC and EML4-ALK fusion. In someembodiments, FISH is used to detect or test for one or more of thefollowing genes, including without limitation: EGFR, SPARC, C-kit, ER,PR, AR, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, TS,HER2, or TOPO2A. In some embodiments, FISH is used to detect or test forone or more of EML4-ALK fusion and IGF-1R. In some embodiments, FISH isused to detect or test various biomarkers, including without limitationone or more of the following: ADA, AR, ASNA, BCL2, BRCA2, c-Met, CD33,CDW52, CES2, DNMT1, EGFR, EML4-ALK fusion, ERBB2, ERCC3, ESR1, FOLR2,GART, GSTP1, HDAC1, hENT-1, HIF1A, HSPCA, IGF-1R, IL2RA, KIT, MLH1, MMR,MS4A1, MASH2, NFKB2, NFKBIA, OGFR, p16, p21, p27, PARP-1, PI3K, PDGFC,PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1,TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, orZAP70.

In some embodiments, FISH is used to detect or test for HER2, anddepending on the results of the HER2 analysis and other molecularprofiling techniques, additional FISH testing may be performed. Theadditional FISH testing can comprise that of CMYC and/or TOP2A. Forexample, FISH testing may indicate that a cancer is HER2+. The cancermay be a breast cancer. HER2+ cancers may then be followed up by FISHtesting for CMYC and TOP2A, whereas HER2− cancers are followed up withFISH testing for CMYC. For some cancers, e.g., triple negative breastcancer (i.e., ER−/PR−/HER2−), additional FISH testing may not beperformed. The decision whether to perform additional FISH testing canbe guided by whether the additional FISH testing is likely to revealinformation about candidate therapies for the cancer. The additionalFISH can be selected on the basis of molecular characteristics of thetumor so that FISH is only performed where it is likely to indicate acandidate therapy for treating the cancer. As described herein, themolecular characteristics of the tumor determined can be determined byone or more of IHC, FISH, DNA microarray and sequence analysis. Thegenes and/or gene products used for FISH analysis can be at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 orall of the genes and/or gene products listed in Table 2. The cancer canbe an ovarian cancer. The cancer can be a CUPS.

In some embodiments, the genes used for the mutation profiling compriseone or more of PIK3CA, EGFR, cKIT, KRAS, NRAS and BRAF. Mutationprofiling can be determined by sequencing, including Sanger sequencing,array sequencing, pyrosequencing, NextGen sequencing, etc. Sequenceanalysis may reveal that genes harbor activating mutations so that drugsthat inhibit activity are indicated for treatment. Alternately, sequenceanalysis may reveal that genes harbor mutations that inhibit oreliminate activity, thereby indicating treatment for compensatingtherapies. In embodiments, sequence analysis comprises that of exon 9and 11 of c-KIT. Sequencing may also be performed on EGFR-kinase domainexons 18, 19, 20, and 21. Mutations, amplifications or misregulations ofEGFR or its family members are implicated in about 30% of all epithelialcancers. Sequencing can also be performed on PI3K, encoded by the PIK3CAgene. This gene is a found mutated in many cancers. Sequencing analysiscan also comprise assessing mutations in one or more ABCC1, ABCG2, ADA,AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR,DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1,FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1,IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1,MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, NRAS, OGFR, PARP1, PDGFC, PDGFRA,PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2,RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5,TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, andZAP70. One or more of the following genes can also be assessed bysequence analysis: ALK, EML4, hENT-1, IGF-1R, HSP90AA1, MMR, p16, p21,p27, PARP-1, PI3K and TLE3. The genes and/or gene products used formutation or sequence analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genesand/or gene products listed in Table 2. The cancer can be an ovariancancer. The cancer can be a CUPS.

In some embodiments, mutational analysis is performed on PIK3CA. Thedecision whether to perform mutational analysis on PIK3CA can be guidedby whether this testing is likely to reveal information about candidatetherapies for the cancer. The PIK3CA mutational analysis can be selectedon the basis of molecular characteristics of the tumor so that theanalysis is only performed where it is likely to indicate a candidatetherapy for treating the cancer. As described herein, the molecularcharacteristics of the tumor determined can be determined by one or moreof IHC, FISH, DNA microarray and sequence analysis. In one embodiment,PIK3CA is analyzed for a HER2+ cancer. The cancer can be a breastcancer. The cancer can be an ovarian cancer. The cancer can be CUPS.

In an aspect, the invention provides a method of identifying a candidatetreatment for a subject in need thereof by using molecular profiling ofsets of known biomarkers. For example, the method can identify achemotherapeutic agent for an individual with a cancer. The methodcomprises: obtaining a sample from the subject; performing animmunohistochemistry (IHC) analysis on the sample to determine an IHCexpression profile on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore, of: SPARC, PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A,TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, MRP1, c-Met, EML4-ALK fusion,hENT-1, IGF-1R, MMR, p16, p21, p27, PARP-1, PI3K, COX2 and TLE3;performing a microarray analysis on the sample to determine a microarrayexpression profile on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore, of: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33,CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2,ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK,HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK,LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1,PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA,RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3,SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA,VHL, YES1, and ZAP70; performing a fluorescent in-situ hybridization(FISH) analysis on the sample to determine a FISH mutation profile on atleast one of EGFR, HER2, EML4-ALK fusion and IGF-1R; performing DNAsequencing or PCR on the sample to determine a sequencing mutationprofile on at least one of KRAS, BRAF, c-KIT, PI3K (PIK3CA), NRAS andEGFR. The method can further comprise comparing the IHC expressionprofile, microarray expression profile, FISH mutation profile andsequencing mutation profile against a rules database, wherein the rulesdatabase comprises a mapping of treatments whose biological activity isknown against diseased cells that: i) overexpress or underexpress one ormore proteins included in the IHC expression profile; ii) overexpress orunderexpress one or more genes included in the microarray expressionprofile; iii) have zero or more mutations in one or more genes includedin the FISH mutation profile; and/or iv) have zero or more mutations inone or more genes included in the sequencing mutation profile; andidentifying the treatment if the comparison against the rules databaseindicates that the treatment should have biological activity against thedisease; and the comparison against the rules database does notcontraindicate the treatment for treating the disease. The disease canbe a cancer. The molecular profiling steps can be performed in anyorder. In some embodiments, not all of the molecular profiling steps areperformed. As a non-limiting example, microarray analysis is notperformed if the sample quality does not meet a threshold value, asdescribed herein. In some embodiments, the IHC expression profiling isperformed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ofthe gene products above. In some embodiments, the microarray expressionprofiling is performed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% of the genes listed above. In some embodiments, the IHCexpression profiling is performed on all of the gene products above. Insome embodiments, the microarray profiling is performed on all of thegenes listed above. In some embodiments, the FISH profiling is performedon all of the gene products above. In some embodiments, the sequenceprofiling is performed on all of the genes listed above.

In a related aspect, the invention provides a method of identifying acandidate treatment for a subject in need thereof by using molecularprofiling of defined sets of known biomarkers. For example, the methodcan identify a chemotherapeutic agent for an individual with a cancer.The method comprises: obtaining a sample from the subject, wherein thesample comprises formalin-fixed paraffin-embedded (FFPE) tissue or freshfrozen tissue, and wherein the sample comprises cancer cells; performingan immunohistochemistry (IHC) analysis on the sample to determine an IHCexpression profile on at least: SPARC, PGP, Her2/neu, ER, PR, c-kit, AR,CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, MRP1,c-Met, EML4-ALK fusion, hENT-1, IGF-1R, MMR, p16, p21, p27, PARP-1,PI3K, and TLE3; performing a microarray analysis on the sample todetermine a microarray expression profile on at least: ABCC1, ABCG2,ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK,DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3,ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1,IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLHEMS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB,PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B,RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1,TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70;performing a fluorescent in-situ hybridization (FISH) analysis on thesample to determine a FISH mutation profile on at least one of EGFR,HER2, EML4-ALK fusion and IGF-1R; performing DNA sequencing on thesample to determine a sequencing mutation profile or PCR on at leastKRAS, BRAF, c-KIT, PI3K (PIK3CA), NRAS and EGFR. The IHC expressionprofile, microarray expression profile, FISH mutation profile andsequencing mutation profile are compared against a rules database,wherein the rules database comprises a mapping of treatments whosebiological activity is known against diseased cells that: i) overexpressor underexpress one or more proteins included in the IHC expressionprofile; ii) overexpress or underexpress one or more genes included inthe microarray expression profile; iii) have zero or more mutations inone or more genes included in the FISH mutation profile; or iv) havezero or more mutations in one or more genes included in the sequencingmutation profile; and identifying the treatment if the comparisonagainst the rules database indicates that the treatment should havebiological activity against the disease; and the comparison against therules database does not contraindicate the treatment for treating thedisease. The disease can be a cancer. The molecular profiling steps canbe performed in any order. In some embodiments, not all of the molecularprofiling steps are performed. As a non-limiting example, microarrayanalysis is not performed if the sample quality does not meet athreshold value, as described herein. In some embodiments, thebiological material is mRNA and the quality control test comprises aA260/A280 ratio and/or a Ct value of RT-PCR using a housekeeping gene,e.g., RPL13a. In embodiments, the mRNA does not pass the quality controltest if the A260/A280 ratio <1.5 or the RPL13a Ct value is >30. In thatcase, microarray analysis may not be performed. Alternately, microarrayresults may be attenuated, e.g., given a lower priority as compared tothe results of other molecular profiling techniques.

In an aspect, the invention provides a method of identifying a candidatetreatment for a subject in need thereof by using molecular profiling ofsets of known biomarkers. For example, the method can identify achemotherapeutic agent for an individual with a cancer. The methodcomprises: obtaining a sample from the subject; performing animmunohistochemistry (IHC) analysis on the sample to determine an IHCexpression profile on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20 or more, of: AR, BCRP, CAV-1, CD20, CD52, CK 5/6, CK14, CK17, c-kit,CMET, COX-2, Cyclin D1, E-Cad, EGFR, ER, ERCC1, HER-2, IGF1R, Ki67,MGMT, MRP1, P53, p95, PDGFR, PGP, PR, PTEN, RRM1, SPARC, TLE3, TOPO1,TOPO2A, TS, TUBB3; performing a microarray analysis on the sample todetermine a microarray expression profile on one or more, e.g. 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more, of: ABCC1, ABCG2,ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, cKit,c-MYC, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERCC1,ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1,HER2/ERBB2, HIF1A, HSP90, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, LCK, LYN,MET, MGMT, MLHE MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC,PDGFRa, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1,RRM2, RRM2B, RXRB, RXRG, SIK2, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5,SPARC, TK1, TNF, TOP2B, TOP2A, TOPO1, TXNRD1, TYMS, VDR, VEGFA, VHL,YES1, and ZAP70; performing a fluorescent in-situ hybridization (FISH)analysis on the sample to determine a FISH mutation profile on at leastone of ALK, cMET, c-MYC, EGFR, HER-2, PIK3CA, and TOPO2A; performing DNAsequencing or PCR on the sample to determine a sequencing mutationprofile on at least one of BRAF, c-kit, EGFR, KRAS, NRAS, and PIK3CA.The method can further comprise comparing the IHC expression profile,microarray expression profile, FISH mutation profile and sequencingmutation profile against a rules database, wherein the rules databasecomprises a mapping of treatments whose biological activity is knownagainst diseased cells that: i) overexpress or underexpress one or moreproteins included in the IHC expression profile; ii) overexpress orunderexpress one or more genes included in the microarray expressionprofile; iii) have zero or more mutations in one or more genes includedin the FISH mutation profile; and/or iv) have zero or more mutations inone or more genes included in the sequencing mutation profile; andidentifying the treatment if the comparison against the rules databaseindicates that the treatment should have biological activity against thedisease; and the comparison against the rules database does notcontraindicate the treatment for treating the disease. The disease canbe a cancer, such as an ovarian cancer, a CUPS, or any other cancerdisclosed herein. The molecular profiling steps can be performed in anyorder. In some embodiments, not all of the molecular profiling steps areperformed. As a non-limiting example, microarray analysis is notperformed if the sample quality does not meet a threshold value, asdescribed herein. In some embodiments, the IHC expression profiling isperformed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ofthe gene products above. In some embodiments, the microarray expressionprofiling is performed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% of the genes listed above. In some embodiments, the IHCexpression profiling is performed on all of the gene products above. Insome embodiments, the microarray profiling is performed on all of thegenes listed above. In some embodiments, the FISH profiling is performedon all of the gene products above. In some embodiments, the sequenceprofiling is performed on all of the genes listed above.

In a related aspect, the invention provides a method of identifying acandidate treatment for a subject in need thereof by using molecularprofiling of defined sets of known biomarkers. For example, the methodcan identify a chemotherapeutic agent for an individual with a cancer.The method comprises: obtaining a sample from the subject, wherein thesample comprises formalin-fixed paraffin-embedded (FFPE) tissue or freshfrozen tissue, and wherein the sample comprises cancer cells; performingan immunohistochemistry (IHC) analysis on the sample to determine an IHCexpression profile on at least: AR, BCRP, CAV-1, CD20, CD52, CK 5/6,CK14, CK17, c-kit, CMET, COX-2, Cyclin D1, E-Cad, EGFR, ER, ERCC1,HER-2, IGF1R, Ki67, MGMT, MRP1, P53, p95, PDGFR, PGP, PR, PTEN, RRM1,SPARC, TLE3, TOPO1, TOPO2A, TS, TUBB3; performing a microarray analysison the sample to determine a microarray expression profile on at least:ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA,CES2, cKit, c-MYC, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2,ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1,HER2/ERBB2, HIF1A, HSP90, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, LCK, LYN,MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC,PDGFRa, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1,RRM2, RRM2B, RXRB, RXRG, SIK2, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5,SPARC, TK1, TNF, TOP2B, TOP2A, TOPO1, TXNRD1, TYMS, VDR, VEGFA, VHL,YES1, and ZAP70; performing a fluorescent in-situ hybridization (FISH)analysis on the sample to determine a FISH mutation profile on at leastone of ALK, cMET, c-MYC, EGFR, HER-2, PIK3CA, and TOPO2A; performing DNAsequencing or PCR on the sample to determine a sequencing mutationprofile on at least BRAF, c-kit, EGFR, KRAS, NRAS, and PIK3CA. The IHCexpression profile, microarray expression profile, FISH mutation profileand sequencing mutation profile are compared against a rules database,wherein the rules database comprises a mapping of treatments whosebiological activity is known against diseased cells that: i) overexpressor underexpress one or more proteins included in the IHC expressionprofile; ii) overexpress or underexpress one or more genes included inthe microarray expression profile; iii) have zero or more mutations inone or more genes included in the FISH mutation profile; or iv) havezero or more mutations in one or more genes included in the sequencingmutation profile; and identifying the treatment if the comparisonagainst the rules database indicates that the treatment should havebiological activity against the disease; and the comparison against therules database does not contraindicate the treatment for treating thedisease. The disease can be a cancer, such as an ovarian cancer, a CUPS,or any other cancer disclosed herein. The molecular profiling steps canbe performed in any order. In some embodiments, not all of the molecularprofiling steps are performed. As a non-limiting example, microarrayanalysis is not performed if the sample quality does not meet athreshold value, as described herein. In some embodiments, thebiological material is mRNA and the quality control test comprises aA260/A280 ratio and/or a Ct value of RT-PCR using a housekeeping gene,e.g., RPL13a. In embodiments, the mRNA does not pass the quality controltest if the A260/A280 ratio <1.5 or the RPL13a Ct value is >30. In thatcase, microarray analysis may not be performed. Alternately, microarrayresults may be attenuated, e.g., given a lower priority as compared tothe results of other molecular profiling techniques.

In some embodiments, molecular profiling is always performed on certaingenes or gene products, whereas the profiling of other genes or geneproducts is optional. For example, IHC expression profiling may beperformed on at least SPARC, TOP2A and/or PTEN. Similarly, microarrayexpression profiling may be performed on at least CD52. In otherembodiments, genes in addition to those listed above are used toidentify a treatment. For example, the group of genes used for the IHCexpression profiling can further comprise DCK, EGFR, BRCA1, CK 14, CK17, CK 5/6, E-Cadherin, p95, PARP-1, SPARC and TLE3. In someembodiments, the group of genes used for the IHC expression profilingfurther comprises Cox-2 and/or Ki-67. In some embodiments, HSPCA isassayed by microarray analysis. In some embodiments, FISH mutation isperformed on c-Myc and TOP2A. In some embodiments, sequencing isperformed on PI3K.

The methods of the invention can be used in any setting whereindifferential expression or mutation analysis have been linked toefficacy of various treatments. In some embodiments, the methods areused to identify candidate treatments for a subject having a cancer.Under these conditions, the sample used for molecular profilingpreferably comprises cancer cells. The percentage of cancer in a samplecan be determined by methods known to those of skill in the art, e.g.,using pathology techniques. Cancer cells can also be enriched from asample, e.g., using microdissection techniques or the like. A sample maybe required to have a certain threshold of cancer cells before it isused for molecular profiling. The threshold can be at least about 5, 10,20, 30, 40, 50, 60, 70, 80, 90 or 95% cancer cells. The threshold candepend on the analysis method. For example, a technique that revealsexpression in individual cells may require a lower threshold that atechnique that used a sample extracted from a mixture of differentcells. In some embodiments, the diseased sample is compared to a normalsample taken from the same patient, e.g., adjacent but non-cancertissue.

In embodiments, the methods of the invention are used detect genefusions, such as those listed in Table 3. A fusion gene is a hybrid genecreated by the juxtaposition of two previously separate genes. This canoccur by chromosomal translocation or inversion, deletion or viatrans-splicing. The resulting fusion gene can cause abnormal temporaland spatial expression of genes, leading to abnormal expression of cellgrowth factors, angiogenesis factors, tumor promoters or other factorscontributing to the neoplastic transformation of the cell and thecreation of a tumor. For example, such fusion genes can be oncogenic dueto the juxtaposition of: 1) a strong promoter region of one gene next tothe coding region of a cell growth factor, tumor promoter or other genepromoting oncogenesis leading to elevated gene expression, or 2) due tothe fusion of coding regions of two different genes, giving rise to achimeric gene and thus a chimeric protein with abnormal activity. Fusiongenes are characteristic of many cancers, such as those listed in Table3. Once a therapeutic intervention is associated with a fusion, thepresence of that fusion in any type of cancer identifies the therapeuticintervention as a candidate therapy for treating the cancer.

TABLE 3 Fusion Genes and Associated Cancers 5′ Upstream 3′ downstreamFusion Gene Fusion Gene Partner Partner Cancer Lineage ACSL3 ETV1Prostate cancer AKAP9 BRAF Papillary thyroid carcinoma Alpha TFEB Renalcell carcinoma ARHGAP20 BRWD3 B-cell chronic lymphocytic leukemia(B-CLL) ASPSCR1 TFE3 Renal-cell carcinoma ATIC ALK Anaplastic large celllymphoma (ALCL) BCL11B TLX3 T-cell acute lymphoblastic/lymphocyticleukemia (T-ALL) BCL3 MYC B-cell chronic lymphocytic leukemia (B-CLL)BCL7A MYC B-cell chronic lymphocytic leukemia (B-CLL) BCR ABL1 Chronicmyelogenous leukemia (CML) BCR FGFR1 CML-like Myeloproliferativedisorder (MPD) BCR JAK2 Chronic myelogenous leukemia (CML) BCR PDGFRAAtypical CML BIRC3 MALT1 B-cell non Hodgkin lymphoma, MALT-lymphomasBRD4 NUT Poorly differentiated epithelial carcinoma (Aggressive midlinecarcinoma) BRWD3 ARHGAP20 B-cell chronic lymphocytic leukemia (B-CLL)BTG1 MYC B-cell chronic lymphocytic leukemia (B-CLL) CARS ALKInflammatory myofibroblastic tumor CANT1 ETV4 Prostate cancer CBFB MYH11Acute myelogenous leukemia (AML) CCDC6 PDGFRB Philadelphia chr negativeMyeloproliferative disorder (MPD) CCDC6 RET Papillary thyroid carcinomaCCND1 FSTL3 Chronic myelogenous leukemia (CML) CD74 ROS1 Non small celllung carcinoma (NSCLC) CDH11 USP6 Aneurysmal bone cyst CDK6 EVI1 Myeloidleukemia CDK6 MLL Acute lymphoblastic/lymphocytic leukemia (ALL) CDK6TLX3 Acute lymphoblastic/lymphocytic leukemia (ALL) CEP110 FGFR1Myeloproliferative disorder (Myeloproliferative disorder (MPD)) CHCHD7PLAG1 Pleomorphic salivary gland adenomas (PA) (Head and Neck) CHIC2ETV6 Acute myelogenous leukemia (AML) CIITA BCL6 Diffuse large B-celllymphoma (DLBCL) CLTC ALK Diffuse large B-cell lymphoma (DLBCL) CLTCTFE3 Pediatric renal adenocarcinoma C15ORF21 ETV1 Prostate cancer COL1A1PDGFB Dermatofibrosarcoma protuberans COL1A1 USP6 Aneurysmal bone cystCOL1A2 PLAG1 Lipoblastoma CRC1 MAML2 Mucoepidermoid carcinoma CRTC1MAML2 Mucoepidermoid carcinomas, Warthin's tumor CRTC3 MAML2Mucoepidermoid carcinoma CTNNB1 PLAG1 Pleomorphic salivary glandadenomas (PA) (Head and Neck) DDX5 ETV4 Prostate cancer EIF4A2 BCL6Non-Hodgkin lymphoma (NHL) EML1 ABL1 T-cell acutelymphoblastic/lymphocytic leukemia (T-ALL) EML4 ALK Non small cell lungcarcinoma (NSCLC) EPC1 PHF1 Endometrial stromal sarcoma ERC1 RETPapillary thyroid carcinoma ETV6 ABL1 Chronic myelogenous leukemia(CML), Acute myelogenous leukemia (AML), Acute lymphoblastic/lymphocyticleukemia (ALL) ETV6 ABL2 T-cell acute lymphoblastic/lymphocytic leukemia(T-ALL), Acute myelogenous leukemia (AML) ETV6 ACSL6 Polycythemia veraETV6 ARNT Acute myelogenous leukemia (AML) ETV6 CDX2 Acute myelogenousleukemia (AML) ETV6 EVI1 Chronic myelogenous leukemia (CML) ETV6 FGFR3Peripheral T-cell lymphoma ETV6 FLT3 ALL, Myeloproliferative disorder(MPD) ETV6 HLXB9 Acute myelogenous leukemia (AML) ETV6 JAK2 Philadelphiachr negative Myeloproliferative disorder (MPD), B cell malignancies ETV6MDS2 Myelodisplastic syndrome ETV6 MN1 Chronic myelogenous leukemia(CML) ETV6 NTRK3 Secretory breast cancer ETV6 PDGFRB Chronicmyelomonocytic leukemia (CMML) ETV6 PER1 Acute myelogenous leukemia(AML) ETV6 RUNX1 Acute lymphoblastic/lymphocytic leukemia (ALL) ETV6 SYKMyelodisplastic syndrome ETV6 TCBA1 Chronic myelogenous leukemia (CML)ETV6 TTL Acute lymphoblastic/lymphocytic leukemia (ALL) EWSR1 ATF1 Softtissue sarcoma EWSR1 DDIT3 Myxoid liposarcoma EWSR1 ERG Ewing sarcomasEWSR1 ETV1 Ewing sarcomas EWSR1 ETV4 Ewing sarcomas EWSR1 FEV Ewingsarcomas EWSR1 FLI1 Ewing sarcomas EWSR1 NR4A3 Malignant tumor of softtissue origin EWSR1 POU5F1 Undifferentiated bone tumor EWSR1 TEC Ewingsarcomas EWSR1 WT1 Soft tissue sarcoma EWSR1 ZNF278 Small round cellsarcoma EWSR1 ZNF384 Acute lymphoblastic leukemia FGFR1OP FGFR1Stem-cell myeloproliferative disorder characterized by myeloidhyperplasia, T-cell lymphoblastic leukemia/lymphoma and peripheral bloodeosinophilia, and it generally progresses to acute myeloid leukemia;FGFR1OP2 FGFR1 Myeloproliferative disorder (MPD) is characterized bymyeloid hyperplasia, eosinophilia and T-cell or B-cell lymphoblasticlymphoma FHIT HMGA2 Pleomorphic salivary gland adenomas (PA) (Head andNeck) FIP1L1 PDGFRA Hypereosinophilia FLT3 ETV6 HypereosinophiliaFLJ35294 ETV1 Prostate cancer FUS ATF1 Angiomatoid fibrous histiocytoma(AFH) FUS CREB3L1 Fibromyxoid sarcoma FUS CREB3L2 Low-grade fibromyxoidsarcoma (LGFMS) FUS DDIT3 Myxoid liposarcoma FUS DDIT3 The Myxoid/RoundCell Liposarcoma FUS ERG Ewing sarcomas GAPDH BCL6 B-cell non Hodgkinlymphoma (B-NHL), Diffuse large B-cell lymphoma (DLBCL) GOLGA5 RETPapillary thyroid carcinoma GOPC ROS1 Glioblastoma HAS2 PLAG1Lipoblastoma HERV ETV1 Prostate cancer HIP1 PDGFRB Chronicmyelomonocytic leukemia (CMML) HIST1H4I BCL6 B-cell Non-Hodgkin lymphoma(B-NHL) HMGA1 LAMA4 Pulmonary chondroid hamartoma HMGA2 CCNB1IP1 Benignmesenchymal tumors HMGA2 COX6C Uterine leiomyoma HMGA2 CXCR7 LipomaHMGA2 FHIT Pleomorphic salivary gland adenomas (PA) (Head and Neck)HMGA2 LHFP Solitary lipomas HMGA2 LPP Lipoma, parosteal lipoma, andpulmonary chondroid hamartoma HMGA2 NFIB Pleomorphic salivary glandadenomas (PA) (Head and Neck) HMGA2 RAD51L1 Uterine leiomyomata HNRPA2B1ETV1 Prostate cancer HOOK3 RET Papillary thyroid carcinoma HRH4 RETPapillary thyroid carcinoma HSP90AA1 BCL6 B cell Non-Hodgkin lymphoma(B-NHL) HSP90AB1 BCL6 B-cell tumors IGH MYC Burkitt's lymphoma IKZF1BCL6 Diffuse large B-cell lymphoma (DLBCL) IL2 TNFRSF17 T-cell acutelymphoblastic leukemia (T-ALL) IL21R BCL6 Diffuse large B-cell lymphoma(DLBCL) ITK SYK Unspecified peripheral T-cell lymphoma JAZF1 PHF1Endometrial stromal sarcomas JAZF1 SUZ12 endometrial stromal tumors andendometrial stromal sarcoma KIAA1509 PDGFRA Chronic eosinophilicleukemia (CEL) KIAA1618 ALK Anaplastic large-cell lymphoma (ALCL) KLK2ETV4 Prostate cancer KTN1 RET Papillary thyroid carcinoma LCP1 BCL6 NonHodgkin follicular, Burkitt lymphomas LIFR PLAG1 Pleomorphic salivarygland adenomas (PA) (Head and Neck) MALAT1 TFEB Pediatric renal neoplasmMEF2D DAZAP1 Acute myelogenous leukemia (AML) MLL ABI1 acute nonlymphoblastic leukemia MLL AFF1 Acute lymphoblastic/lymphocytic leukemia(ALL), Acute myelogenous leukemia (AML) MLL AFF3 Acutelymphoblastic/lymphocytic leukemia (ALL) MLL AFF4 Acutelymphoblastic/lymphocytic leukemia (ALL) MLL ARHGAP26 Acute monocyticleukemia (Acute myelogenous leukemia (AML) (M5b) MLL ARHGEF12 Acutemyelogenous leukemia (AML) MLL CASC5 Acute myelogenous leukemia (AML)MLL CBL Acute myelogenous leukemia (AML) MLL CLP1 Monoblastic leukemiaMLL CREBBP Acute myelogenous leukemia (AML) MLL CXXC6 Acutelymphoblastic/lymphocytic leukemia (ALL) MLL DAB2IP Acute myelogenousleukemia (AML) MLL ELL Acute myelogenous leukemia (AML) MLL EP300 Acutemyelogenous leukemia (AML) MLL EPS15 Acute myelogenous leukemia (AML)MLL FNBP1 Acute myelogenous leukemia (AML) MLL FOXO3A Acute myelogenousleukemia (AML) MLL GAS7 Acute lymphoblastic/lymphocytic leukemia (ALL)MLL GMPS Acute myelogenous leukemia (AML) MLL GPHN Acute myelogenousleukemia (AML) MLL LASP1 Infant acute myeloid leukemia Acute myelogenousleukemia (AML)-M4 MLL LPP Secondary acute leukemia MLL MAPRE1 Pro-Bacute lymphoblastic leukemia MLL MLL Acute myeloid and lymphoid leukemiaMLL MLLT1 Acute myelogenous leukemia (AML) MLL MLLT10 Pediatric acutemegakaryoblastic leukemia AND acute monoblastic leukemia MLL MLLT11Acute myelogenous leukemia (AML) MLL MLLT3 Acute myelogenous leukemia(AML) MLL MLLT4 M4/M5 ANLL MLL MLLT6 Acute myelogenous leukemia (AML)MLL MLLT7 Acute leukemias MLL MYO1F Acute myelogenous leukemia (AML) MLLPICALM Acute myelogenous leukemia (AML) MLL RARA M5 acute nonlymphocytic leukemia (ANLL) MLL SEPT11 Chronic neutrophilic leukemia MLLSEPT2 Acute myelogenous leukemia (AML), therapy- related myelodysplasticsyndrome MLL SEPT5 De novo acute non lymphocytic leukemia MLL SEPT6Acute myelogenous leukemia (AML) MLL SEPT9 Myeloid neoplasia MLL SH3GL1Acute leukemia MLL SORBS2 Acute myelogenous leukemia (AML) MLL ZFYVE19Acute myelogenous leukemia (AML) MSI2 HOXA9 Chronic myelogenous leukemia(CML) MSN ALK Anaplastic large cell lymphoma (ALCL) MYC BCL7A High-gradeB cell Non-Hodgkin lymphoma (NHL) MYC BTG1 B-cell chronic lymphocyticleukemia (B-CLL) MYH9 ALK Anaplastic large cell lymphoma (ALCL) MYST3ASXL2 Therapy-related myelodysplastic syndrome MYST3 CREBBP Acutemyelogenous leukemia (AML) MYST3 EP300 Acute myelomonocytic or monocyticleukemia (M4 or M5 Acute myelogenous leukemia (AML)) MYST3 NCOA2 Acuteleukemia MYST4 CREBBP Acute myelogenous leukemia (AML) NACA BCL6Non-Hodgkin lymphoma (NHL) NCOA4 RET Papillary thyroid carcinoma NINPDGFRB Chronic myeloproliferative disorder with eosinophilia NONO TFE3Renal cell carcinoma NPM1 ALK Anaplastic large-cell lymphomas (ALCL)NPM1 MLF1 Acute myelogenous leukemia (AML) NPM1 RARA Acute promyelocyticleukemia (APML) NUMA1 RARA Atypical M3 acute non lymphoblastic leukemia(ANLL) NUP214 ABL1 T-cell acute lymphoblastic/lymphocytic leukemia(T-ALL) NUP214 DEK Acute myelogenous leukemia (AML) and myelodysplasticsyndrome NUP214 SET Acute undifferentiated leukemia (AUL) NUP98 ADD3T-cell acute lymphoblastic leukemia with biphenotypic characteristics(T/myeloid) NUP98 CCDC28A Acute megakaryoblastic leukemia, AND T cellacute lymphoblastic leukemia (T-ALL) NUP98 DDX10 De novo or secondarymyeloid malignancies NUP98 HOXA11 Juvenile myelomonocytic leukemia(JMML) NUP98 HOXA13 Acute myelogenous leukemia (AML) NUP98 HOXA9 Acutemyelogenous leukemia (AML) NUP98 HOXC11 Acute myelogenous leukemia (AML)NUP98 HOXC13 Acute myelogenous leukemia (AML) NUP98 HOXD11 Acutemyelomonocytic leukemia NUP98 HOXD13 Acute myelogenous leukemia (AML)NUP98 JARID1A Acute leukemia NUP98 NSD1 Childhood acute myelogenousleukemia (AML) NUP98 PRRX1 M2-ANLL, Non Hodgkin lymphoma (NHL) NUP98PRRX2 Acute myelogenous leukemia (AML) NUP98 PSIP1 Acute nonlymphoblastic leukemia NUP98 RAP1GDS1 T acute lymphoblastic leukemiaNUP98 TOP1 Acute myelogenous leukemia (AML) NUP98 WHSC1L1 Acutemyelogenous leukemia (AML) NUT BRD4 Midline carcinoma OMD USP6Aneurysmal bone cyst PAX3 FOXO1 Rhabdomyosarcoma PAX5 ETV6 Acutelymphoblastic/lymphocytic leukemia (ALL) PAX7 FOXO1 Alveolarrhabdomyosarcomas PAX8 PPARy Follicular thyroid carcinoma PCM1 JAK2Myeloproliferative disorder (MPD) and acute erythroid leukemia PCM1 RETPapillary thyroid carcinoma PDE4DIP PDGFRB Chronic eosinophilic leukemia(CEL) PICALM MLLT10 CML, Acute myelogenous leukemia (AML) PIM1 BCL6Diffuse large B-cell lymphoma (DLBCL) PML RARA Acute promyelocyticleukemia (APML) POU2AF1 BCL6 Non-Hodgkin lymphoma (NHL) PRCC TFE3 Renalcell carcinoma PRDM16 EVI1 MDS and Acute myelogenous leukemia (AML)PRKAR1A RET Papillary thyroid carcinoma RABEP1 PDGFRB Myeloproliferativedisorder (MPD) and Acute myelogenous leukemia (AML), RANBP2 ALKInflammatory myofibroblastic tumors (IMT) RBM15 MKL1 Acute myelogenousleukemia (AML) RFG RET Papillary thyroid carcinoma RFG9 RET Papillarythyroid carcinoma RHOH BCL6 Follicular centrocytic-centroblasticlymphoma. Ria RET Papillary thyroid carcinoma RLF MYCL1 Small-cell lungcancer (SCLC) RPN1 EVI1 Acute non lymphocytic leukemia (ANLL),Myelodysplastic syndrome RUNX1 CBFA2T3 Myeloid malignancies. RUNX1 EVI1Acute myelogenous leukemia (AML), therapy- related MDS and chronicmyeloid leukemia in blastic phase RUNX1 MDS1 Acute myelogenous leukemia(AML), therapy- related MDS and chronic myeloid leukemia in blasticphase RUNX1 RPL22 Acute myelogenous leukemia (AML) RUNX1 RUNX1T1 Acutemyelogenous leukemia (AML) RUNX1 SH3D19 Acute myelogenous leukemia (AML)RUNX1 USP42 Acute myelogenous leukemia (AML) RUNX1 YTHDF2 Acutemyelogenous leukemia (AML) RUNX1 ZNF687 Acute myelogenous leukemia (AML)SEC31A ALK Diffuse large B-cell lymphoma (DLBCL) SENP6 TCBA1 T-celllymphoma SFPQ TFE3 Renal cell carcinoma SFRS3 BCL6 Follicular lymphomaSLC5A3 ERG Prostate cancer SLC45A3 ETV1 Prostate cancer SLC45A3 ETV5Prostate cancer SPECC1 PDGFRB Juvenile myelomonocytic leukemia SS18 SSX1Synovial sarcoma SS18 SSX2 Synovial sarcoma SS18 SSX4 Synovial sarcomaSS18L1 SSX1 Synovial sarcoma STAT5B RARA Acute promyelocytic leukemia(APML) TAF15 NR4A3 Ewing's sarcoma/primitive neuroectodermal tumor TAF15TEC Ewing sarcomas TAF15 ZNF384 Acute myelogenous leukemia (AML) TAL1STIL T-cell malignancies (T-ALL) TCBA1 ETV6 Acutelymphoblastic/lymphocytic leukemia (ALL) TCEA1 PLAG1 Pleomorphicsalivary gland adenomas (PA) (Head and Neck) TCF12 NR4A3 Extraskeletalmyxoid chondrosarcoma TCF12 TEC Extraskeletal myxoid chondrosarcoma TCF3HLF pre-B-cell acute lymphoblastic leukemia TCF3 PBX1 Acutelymphoblastic/lymphocytic leukemia (ALL) TCF3 TFPT Acutelymphoblastic/lymphocytic leukemia (ALL) TFG ALK Anaplastic large celllymphoma (ALCL), Non small cell lung carcinoma (NSCLC) TFG NR4A3Extraskeletal myxoid chondrosarcoma TFG NTRK1 Papillary thyroidcarcinoma TFRC BCL6 B-cell non Hodgkin lymphoma (B-NHL), Diffuse largeB-cell lymphoma (DLBCL) THRAP3 USP6 Aneurysmal bone cysts TIAF1 FGFR1Myeloproliferative disorder (MPD) TMPRSS2 ERG Prostate cancer TMPRSS2ETV1 Prostate cancer TMPRSS2 ETV4 Prostate cancer TMPRSS2 ETV5 Prostatecancer TP53BP1 PDGFRB CML-like disorder associated with eosinophiliaTPM3 ALK Anaplastic large cell lymphoma (ALCL) TPM3 NTRK1 Papillarythyroid carcinoma TPM3 PDGFRB Chronic eosinophilic leukemia (CEL) TPM3TPR Papillary thyroid carcinoma TPM4 ALK Inflammatory MyofibroblasticTumors TPR MET Papillary thyroid carcinoma TPR NTRK1 Papillary thyroidcarcinoma TRIM24 FGFR1 Myeloproliferative disorder (MPD) TRIM24 RARAMyeloproliferative disorder (MPD) TRIM24 RET Papillary thyroid carcinomaTRIM27 RET Papillary thyroid carcinoma TRIM33 RET Papillary thyroidcarcinoma TRIP11 PDGFRB Acute myelogenous leukemia (AML) TTL ETV6 Acutelymphoblastic/lymphocytic leukemia (ALL) ZBTB16 RARA Acute promyelocyticleukemia (APML) ZMYM2 FGFR1 Stem cell leukemia lymphoma syndrome (SCLL)

The presence of fusion genes, e.g., those described in Table 3 orelsewhere herein, can be used to guide therapeutic selection. Forexample, the BCR-ABL gene fusion is a characteristic molecularaberration in ˜90% of chronic myelogenous leukemia (CML) and in a subsetof acute leukemias (Kurzrock et al., Annals of Internal Medicine 2003;138:819-830). The BCR-ABL results from a translocation betweenchromosomes 9 and 22, commonly referred to as the Philadelphiachromosome or Philadelphia translocation. The translocation bringstogether the 5′ region of the BCR gene and the 3′ region of ABL1,generating a chimeric BCR-ABL1 gene, which encodes a protein withconstitutively active tyrosine kinase activity (Mittleman et al., NatureReviews Cancer 2007; 7:233-245). The aberrant tyrosine kinase activityleads to de-regulated cell signaling, cell growth and cell survival,apoptosis resistance and growth factor independence, all of whichcontribute to the pathophysiology of leukemia (Kurzrock et al., Annalsof Internal Medicine 2003; 138:819-830). Patients with the Philadelphiachromosome are treated with imatinib and other targeted therapies.Imatinib binds to the site of the constitutive tyrosine kinase activityof the fusion protein and prevents its activity. Imatinib treatment hasled to molecular responses (disappearance of BCR-ABL+ blood cells) andimproved progression-free survival in BCR-ABL+ CML patients (Kantarjianet al., Clinical Cancer Research 2007; 13:1089-1097).

Another fusion gene, IGH-MYC, is a defining feature of ˜80% of Burkitt'slymphoma (Ferry et al. Oncologist 2006; 11:375-83). The causal event forthis is a translocation between chromosomes 8 and 14, bringing the c-Myconcogene adjacent to the strong promoter of the immunoglobulin heavychain gene, causing c-myc overexpression (Mittleman et al., NatureReviews Cancer 2007; 7:233-245). The c-myc rearrangement is a pivotalevent in lymphomagenesis as it results in a perpetually proliferativestate. It has wide ranging effects on progression through the cellcycle, cellular differentiation, apoptosis, and cell adhesion (Ferry etal. Oncologist 2006; 11:375-83).

A number of recurrent fusion genes have been catalogued in the Mittlemandatabase (cgap.nci.nih.gov/Chromosomes/Mitelman) The gene fusions can beused to characterize neoplasms and cancers and guide therapy using thesubject methods described herein. For example, TMPRSS2-ERG, TMPRSS2-ETVand SLC45A3-ELK4 fusions can be detected to characterize prostatecancer; and ETV6-NTRK3 and ODZ4-NRG1 can be used to characterize breastcancer. The EML4-ALK, RLF-MYCL1, TGF-ALK, or CD74-ROS1 fusions can beused to characterize a lung cancer. The ACSL3-ETV1, C150RF21-ETV1,FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5,SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4 fusions can be used tocharacterize a prostate cancer. The GOPC-ROS1 fusion can be used tocharacterize a brain cancer. The CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2,HMGA2-NFIB, LIFR-PLAG1, or TCEA1-PLAG1 fusions can be used tocharacterize a head and neck cancer. The ALPHA-TFEB, NONO-TFE3,PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3, or MALAT1-TFEB fusions can be used tocharacterize a renal cell carcinoma (RCC). The AKAP9-BRAF, CCDC6-RET,ERC1-RETM, GOLGA5-RET, HOOK3-RET, HRH4-RET, KTN1-RET, NCOA4-RET,PCM1-RET, PRKARA1A-RET, RFG-RET, RFG9-RET, Ria-RET, TGF-NTRK1,TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR-NTRK1, TRIM24-RET, TRIM27-RET orTRIM33-RET fusions can be used to characterize a thyroid cancer and/orpapillary thyroid carcinoma; and the PAX8-PPARy fusion can be analyzedto characterize a follicular thyroid cancer. Fusions that are associatedwith hematological malignancies include without limitation TTL-ETV6,CDK6-MLL, CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1, ETV6-TTL, MLL-AFF1,MLL-AFF3, MLL-AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX1 or TCF3-TFPT, whichare characteristic of acute lymphocytic leukemia (ALL); BCL11B-TLX3,IL2-TNFRF S17, NUP214-ABL1, NUP98-CCDC28A, TAL1-STIL, or ETV6-ABL2,which are characteristic of T-cell acute lymphocytic leukemia (T-ALL);ATIC-ALK, KIAA1618-ALK, MSN-ALK, MYH9-ALK, NPM1-ALK, TGF-ALK orTPM3-ALK, which are characteristic of anaplastic large cell lymphoma(ALCL); BCR-ABL1, BCR-JAK2, ETV6-EVI1, ETV6-MN1 or ETV6-TCBA1,characteristic of chronic myelogenous leukemia (CML); CBFB-MYH11,CHIC2-ETV6, ETV6-ABL1, ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HDCB9,ETV6-PER1, MEF2D-DAZAP1, AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12,MLL-CASC5, MLL-CBL, MLL-CREBBP, MLL-DAB21P, MLL-ELL, MLL-EP300,MLL-EPS15, MLL-FNBP1, MLL-FOXO3A, MLL-GMPS, MEL-GPHN, MLL-MLLT1,MLL-MLLT11, MLL-MLLT3, MLL-MLLT6, MLL-MYO1F, MLL-PICALM, MLL-SEPT2,MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2, MYST-CREBBP, NPM1-MLF1,NUP98-HOXA13, PRDM16-EVI1, RABEP1-PDGFRB, RUNX1-EVI1, RUNX1-MDS1,RUNX1-RPL22, RUNX1-RUNX1T1, RUNX1-SH3D19, RUNX1-USP42, RUNX1-YTHDF2,RUNX1-ZNF687, or TAF15-ZNF-384, which are characteristic of acutemyeloid leukemia (AML); CCND1-FSTL3, which is characteristic of chroniclymphocytic leukemia (CLL); BCL3-MYC, MYC-BTG1, BCL7A-MYC,BRWD3-ARHGAP20 or BTG1-MYC, which are characteristic of B-cell chroniclymphocytic leukemia (B-CLL); CITTA-BCL6, CLTC-ALK, IL21R-BCL6,PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, which are characteristicof diffuse large B-cell lymphomas (DLBCL); FLIP1-PDGFRA, FLT3-ETV6,KIAA1509-PDGFRA, PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB, orTPM3-PDGFRB, which are characteristic of hyper eosinophilia/chroniceosinophilia; and IGH-MYC or LCP1-BCL6, which are characteristic ofBurkitt's lymphoma. One of skill will understand that additionalfusions, including those yet to be identified to date, can be used toguide treatment once their presence is associated with a therapeuticintervention.

The fusion genes and gene products can be detected using one or moretechniques described herein. In some embodiments, the sequence of thegene or corresponding mRNA is determined, e.g., using Sanger sequencing,NextGen sequencing, pyrosequencing, DNA microarrays, etc. Chromosomalabnormalities can be assessed using FISH or PCR techniques, amongothers. For example, a break apart probe can be used for FISH detectionof ALK fusions such as EML4-ALK, KIF5B-ALK and/or TFG-ALK. As analternate, PCR can be used to amplify the fusion product, whereinamplification or lack thereof indicates the presence or absence of thefusion, respectively. In some embodiments, the fusion protein fusion isdetected. Appropriate methods for protein analysis include withoutlimitation mass spectroscopy, electrophoresis (e.g., 2D gelelectrophoresis or SDS-PAGE) or antibody related techniques, includingimmunoassay, protein array or immunohistochemistry. The techniques canbe combined. As a non-limiting example, indication of an ALK fusion byFISH can be confirmed for ALK expression using IHC, or vice versa.

Treatment Selection

The systems and methods allow identification of one or more therapeutictargets whose projected efficacy can be linked to therapeutic efficacy,ultimately based on the molecular profiling. Illustrative schemes forusing molecular profiling to identify a treatment regime are shown inFIGS. 2, 39 and 42, each of which is described in further detail herein.The invention comprises use of molecular profiling results to suggestassociations with treatment responses. In an embodiment, the appropriatebiomarkers for molecular profiling are selected on the basis of thesubject's tumor type. These suggested biomarkers can be used to modify adefault list of biomarkers. In other embodiments, the molecularprofiling is independent of the source material. In some embodiments,rules are used to provide the suggested chemotherapy treatments based onthe molecular profiling test results. In an embodiment, the rules aregenerated from abstracts of the peer reviewed clinical oncologyliterature. Expert opinion rules can be used but are optional. In anembodiment, clinical citations are assessed for their relevance to themethods of the invention using a hierarchy derived from the evidencegrading system used by the United States Preventive Services Taskforce.The “best evidence” can be used as the basis for a rule. The simplestrules are constructed in the format of “if biomarker positive thentreatment option one, else treatment option two.” Treatment optionscomprise no treatment with a specific drug, treatment with a specificdrug or treatment with a combination of drugs. In some embodiments, morecomplex rules are constructed that involve the interaction of two ormore biomarkers. In such cases, the more complex interactions aretypically supported by clinical studies that analyze the interactionbetween the biomarkers included in the rule. Finally, a report can begenerated that describes the association of the chemotherapy responseand the biomarker and a summary statement of the best evidencesupporting the treatments selected. Ultimately, the treating physicianwill decide on the best course of treatment.

As a non-limiting example, molecular profiling might reveal that theEGFR gene is amplified or overexpressed, thus indicating selection of atreatment that can block EGFR activity, such as the monoclonal antibodyinhibitors cetuximab and panitumumab, or small molecule kinaseinhibitors effective in patients with activating mutations in EGFR suchas gefitinib, erlotinib, and lapatinib. Other anti-EGFR monoclonalantibodies in clinical development include zalutumumab, nimotuzumab, andmatuzumab. The candidate treatment selected can depend on the settingrevealed by molecular profiling. For example, kinase inhibitors areoften prescribed with EGFR is found to have activating mutations.Continuing with the illustrative embodiment, molecular profiling mayalso reveal that some or all of these treatments are likely to be lesseffective. For example, patients taking gefitinib or erlotinibeventually develop drug resistance mutations in EGFR. Accordingly, thepresence of a drug resistance mutation would contraindicate selection ofthe small molecule kinase inhibitors. One of skill will appreciate thatthis example can be expanded to guide the selection of other candidatetreatments that act against genes or gene products whose differentialexpression is revealed by molecular profiling. Similarly, candidateagents known to be effective against diseased cells carrying certainnucleic acid variants can be selected if molecular profiling revealssuch variants.

As another example, consider the drug imatinib, currently marketed byNovartis as Gleevec in the US in the form of imatinib mesylate. Imatinibis a 2-phenylaminopyrimidine derivative that functions as a specificinhibitor of a number of tyrosine kinase enzymes. It occupies thetyrosine kinase active site, leading to a decrease in kinase activity.Imatinib has been shown to block the activity of Abelson cytoplasmictyrosine kinase (ABL), c-Kit and the platelet-derived growth factorreceptor (PDGFR). Thus, imatinib can be indicated as a candidatetherapeutic for a cancer determined by molecular profiling tooverexpress ABL, c-KIT or PDGFR. Imatinib can be indicated as acandidate therapeutic for a cancer determined by molecular profiling tohave mutations in ABL, c-KIT or PDGFR that alter their activity, e.g.,constitutive kinase activity of ABLs caused by the BCR-ABL mutation. Asan inhibitor of PDGFR, imatinib mesylate appears to have utility in thetreatment of a variety of dermatological diseases.

Cancer therapies that can be identified as candidate treatments by themethods of the invention include without limitation: 13-cis-RetinoicAcid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil,5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®,Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®,Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®,Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine,BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide,BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225,Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine,Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013,CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil,Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11,Cyclophosphamide, Cytadren®, Cytarabine, Cytarabine Liposomal,Cytosar-U®, Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin, Diftitox, DepoCyt™, Dexamethasone, DexamethasoneAcetate Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD,DIC, Diodex Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal,Droxia™, DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™,Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux,Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol Etopophos®,Etoposide, Etoposide Phosphate, Eulexin®, Everolimus, Evista®,Exemestane, Fareston®, Faslodex®, Femara®, Filgrastim, Floxuridine,Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream),Fluoxymesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec™,Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte-Colony StimulatingFactor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®,Herceptin®, Hexadrol, Hexylen®, Hexamethylmelamine, HMM, Hycamtin®,Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone SodiumPhosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate,Hydroxyurea, Ibritumomab, Ibritumomab, Tiuxetan, Idamycin®, Idarubicin,Ifex®, IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, ImidazoleCarboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin −2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®,Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase (t),Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole,Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™,Liposomal Ara-C Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®,Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine,Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®,Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™,Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRONT™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza, Zometa®, and anyappropriate combinations thereof.

The candidate treatments identified according to the subject methods canbe chosen from the class of therapeutic agents identified asAnthracyclines and related substances, Anti-androgens, Anti-estrogens,Antigrowth hormones (e.g., Somatostatin analogs), Combination therapy(e.g., vincristine, bcnu, melphalan, cyclophosphamide, prednisone(VBMCP)), DNA methyltransferase inhibitors, Endocrine therapy—Enzymeinhibitor, Endocrine therapy—other hormone antagonists and relatedagents, Folic acid analogs (e.g., methotrexate), Folic acid analogs(e.g., pemetrexed), Gonadotropin releasing hormone analogs,Gonadotropin-releasing hormones, Monoclonal antibodies(EGFR-Targeted—e.g., panitumumab, cetuximab), Monoclonal antibodies(Her2-Targeted—e.g., trastuzumab), Monoclonal antibodies(Multi-Targeted—e.g., alemtuzumab), Other alkylating agents, Otherantineoplastic agents (e.g., asparaginase), Other antineoplastic agents(e.g., ATRA), Other antineoplastic agents (e.g., bexarotene), Otherantineoplastic agents (e.g., celecoxib), Other antineoplastic agents(e.g., gemcitabine), Other antineoplastic agents (e.g., hydroxyurea),Other antineoplastic agents (e.g., irinotecan, topotecan), Otherantineoplastic agents (e.g., pentostatin), Other cytotoxic antibiotics,Platinum compounds, Podophyllotoxin derivatives (e.g., etoposide),Progestogens, Protein kinase inhibitors (EGFR-Targeted), Protein kinaseinhibitors (Her2 targeted therapy—e.g., lapatinib), Pyrimidine analogs(e.g., cytarabine), Pyrimidine analogs (e.g., fluoropyrimidines),Salicylic acid and derivatives (e.g., aspirin), Src-family proteintyrosine kinase inhibitors (e.g., dasatinib), Taxanes, Taxanes (e.g.,nab-paclitaxel), Vinca Alkaloids and analogs, Vitamin D and analogs,Monoclonal antibodies (Multi-Targeted—e.g., bevacizumab), Protein kinaseinhibitors (e.g., imatinib, sorafenib, sunitinib).

In some embodiments, the candidate treatments identified according tothe subject methods are chosen from at least the groups of treatmentsconsisting of 5-fluorouracil, abarelix, alemtuzumab, aminoglutethimide,anastrozole, asparaginase, aspirin, ATRA, azacitidine, bevacizumab,bexarotene, bicalutamide, calcitriol, capecitabine, carboplatin,celecoxib, cetuximab, chemotherapy, cholecalciferol, cisplatin,cytarabine, dasatinib, daunorubicin, decitabine, doxorubicin,epirubicin, erlotinib, etoposide, exemestane, flutamide, fulvestrant,gefitinib, gemcitabine, gonadorelin, goserelin, hydroxyurea, imatinib,irinotecan, lapatinib, letrozole, leuprolide, liposomal-doxorubicin,medroxyprogesterone, megestrol, megestrol acetate, methotrexate,mitomycin, nab-paclitaxel, octreotide, oxaliplatin, paclitaxel,panitumumab, pegaspargase, pemetrexed, pentostatin, sorafenib,sunitinib, tamoxifen, Taxanes, temozolomide, toremifene, trastuzumab,VBMCP, and vincristine.

Rules Engine

In some embodiments, a database is created that maps treatments andmolecular profiling results. The treatment information can include theprojected efficacy of a therapeutic agent against cells having certainattributes that can be measured by molecular profiling. The molecularprofiling can include differential expression or mutations in certaingenes, proteins, or other biological molecules of interest. Through themapping, the results of the molecular profiling can be compared againstthe database to select treatments. The database can include bothpositive and negative mappings between treatments and molecularprofiling results. In some embodiments, the mapping is created byreviewing the literature for links between biological agents andtherapeutic agents. For example, a journal article, patent publicationor patent application publication, scientific presentation, etc can bereviewed for potential mappings. The mapping can include results of invivo, e.g., animal studies or clinical trials, or in vitro experiments,e.g., cell culture. Any mappings that are found can be entered into thedatabase, e.g., cytotoxic effects of a therapeutic agent against cellsexpressing a gene or protein. In this manner, the database can becontinuously updated. It will be appreciated that the methods of theinvention are updated as well.

The rules can be generated by an evidence-based literature review.Biomarker research is continues to provide a better understanding of theclinical behavior and biology of cancer. This body of literature can bemaintained in an up-to-date data repository incorporating the latestclinical studies relevant to treatment options and potential clinicaloutcomes. The studies can be ranked so that only those with thestrongest or most reliable evidence are selected for rules generation.For example, the rules generation can employ the grading system from thecurrent methods of the U.S. Preventive Services Task Force. Theliterature evidence can be reviewed and evaluated based on the strengthof clinical evidence supporting associations between biomarkers andtreatments in the literature study. This process can be performed by astaff of scientists, physicians and other skilled reviewers. The processcan also be automated in whole or in part by using language search andheuristics to identify relevant literature. The rules can be generatedby a review of a plurality of literature references, e.g., tens,hundreds, thousands or more literature articles.

In another aspect, the invention provides a method of generating a setof evidence-based associations, comprising: (a) searching one or moreliterature database by a computer using an evidence-based medicinesearch filter to identify articles comprising a gene or gene productthereof, a disease, and one or more therapeutic agent; (b) filtering thearticles identified in (a) to compile evidence-based associationscomprising the expected benefit and/or the expected lack of benefit ofthe one or more therapeutic agent for treating the disease given thestatus of the gene or gene product; (c) adding the evidence-basedassociations compiled in (b) to the set of evidence-based associations;and (d) repeating steps (a)-(c) for an additional gene or gene productthereof. The status of the gene can include one or more assessments asdescribed herein which relate to a biological state, e.g., one or moreof an expression level, a copy number, and a mutation. The genes or geneproducts thereof can be one or more genes or gene products thereofselected from Table 2. For example, the method can be repeated for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more of the genes orgene products thereof in Table 2. The genes or gene products thereof canalso comprise all genes or gene products thereof in any one of Table 2,Table 10, Table 11, and Table 12. The disease can be a disease describedhere, e.g., in embodiment the disease comprises an ovarian cancer. Theone or more literature database can be selected from the groupconsisting of the National Library of Medicine's (NLM's) MEDLINE™database of citations, a patent literature database, and a combinationthereof.

Evidence-based medicine (EBM) or evidence-based practice (EBP) aims toapply the best available evidence gained from the scientific method toclinical decision making. This approach assesses the strength ofevidence of the risks and benefits of treatments (including lack oftreatment) and diagnostic tests. Evidence quality can be assessed basedon the source type (from meta-analyses and systematic reviews ofdouble-blind, placebo-controlled clinical trials at the top end, down toconventional wisdom at the bottom), as well as other factors includingstatistical validity, clinical relevance, currency, and peer-reviewacceptance. Evidence-based medicine filters are searches that have beendeveloped to facilitate searches in specific areas of clinical medicinerelated to evidence-based medicine (diagnosis, etiology, meta-analysis,prognosis and therapy). They are designed to retrieve high qualityevidence from published studies appropriate to decision-making. Theevidence-based medicine filter used in the invention can be selectedfrom the group consisting of a generic evidence-based medicine filter, aMcMaster University optimal search strategy evidence-based medicinefilter, a University of York statistically developed searchevidence-based medicine filter, and a University of California SanFrancisco systemic review evidence-based medicine filter. See e.g., USPatent Publication 20080215570; Shojania and Bero. Taking advantage ofthe explosion of systematic reviews: an efficient MEDLINE searchstrategy. Eff Clin Pract. 2001 July-August; 4(4):157-62; Ingui andRogers. Searching for clinical prediction rules in MEDLINE. J Am MedInform Assoc. 2001 July-August; 8(4):391-7; Haynes et al., Optimalsearch strategies for retrieving scientifically strong studies oftreatment from Medline: analytical survey. BMJ. 2005 May 21;330(7501):1179; Wilczynski and Haynes. Consistency and accuracy ofindexing systematic review articles and meta-analyses in medline. HealthInfo Libr J. 2009 September; 26(3):203-10; which references areincorporated by reference herein in their entirety. A generic filter canbe a customized filter based on an algorithm to identify the desiredreferences from the one or more literature database. For example, themethod can use one or more approach as described in U.S. Pat. No.5,168,533 to Kato et al., U.S. Pat. No. 6,886,010 to Kostoff, or USPatent Application Publication No. 20040064438 to Kostoff; whichreferences are incorporated by reference herein in their entirety.

The further filtering of articles identified by the evidence-basedmedicine filter can be performed using a computer, by one or more expertuser, or combination thereof. The one or more expert can be a trainedscientist or physician. In embodiments, the set of evidence-basedassociations comprise one or more of the rules in Table 5. For example,the set of evidence-based associations can include at least 5, 10, 25,50 or 100 rules in Table 5. In some embodiments, the set ofevidence-based associations comprises or consists of all of the rules inTable 5.

In an aspect, the invention provides a computer readable mediumcomprising the set of evidence-based associations generated by thesubject methods. The invention further provides a computer readablemedium comprising one or more rules in Table 5 herein. In an embodiment,the computer readable medium comprises at least 5, 10, 25, 50 or 100rules in Table 5. For example, the computer readable medium can compriseall rules in Table 5.

The rules for the mappings can contain a variety of supplementalinformation. In some embodiments, the database contains prioritizationcriteria. For example, a treatment with more projected efficacy in agiven setting can be preferred over a treatment projected to have lesserefficacy. A mapping derived from a certain setting, e.g., a clinicaltrial, may be prioritized over a mapping derived from another setting,e.g., cell culture experiments. A treatment with strong literaturesupport may be prioritized over a treatment supported by morepreliminary results. A treatment generally applied to the type ofdisease in question, e.g., cancer of a certain tissue origin, may beprioritized over a treatment that is not indicated for that particulardisease. Mappings can include both positive and negative correlationsbetween a treatment and a molecular profiling result. In a non-limitingexample, one mapping might suggest use of a kinase inhibitor likeerlotinib against a tumor having an activating mutation in EGFR, whereasanother mapping might suggest against that treatment if the EGFR alsohas a drug resistance mutation. Similarly, a treatment might beindicated as effective in cells that overexpress a certain gene orprotein but indicated as not effective if the gene or protein isunderexpressed.

The selection of a candidate treatment for an individual can be based onmolecular profiling results from any one or more of the methodsdescribed. Alternatively, selection of a candidate treatment for anindividual can be based on molecular profiling results from more thanone of the methods described. For example, selection of treatment for anindividual can be based on molecular profiling results from FISH alone,IHC alone, or microarray analysis alone. In other embodiments, selectionof treatment for an individual can be based on molecular profilingresults from IHC, FISH, and microarray analysis; IHC and FISH; IHC andmicroarray analysis, or FISH and microarray analysis. Selection oftreatment for an individual can also be based on molecular profilingresults from sequencing or other methods of mutation detection.Molecular profiling results may include mutation analysis along with oneor more methods, such as IHC, immunoassay, and/or microarray analysis.Different combinations and sequential results can be used. For example,treatment can be prioritized according the results obtained by molecularprofiling. In an embodiment, the prioritization is based on thefollowing algorithm: 1) IHC/FISH and microarray indicates same target asa first priority; 2) IHC positive result alone next priority; or 3)microarray positive result alone as last priority. Sequencing can alsobe used to guide selection. In some embodiments, sequencing reveals adrug resistance mutation so that the effected drug is not selected evenif techniques including IHC, microarray and/or FISH indicatedifferential expression of the target molecule. Any suchcontraindication, e.g., differential expression or mutation of anothergene or gene product may override selection of a treatment.

An illustrative listing of microarray expression results versuspredicted treatments is presented in Table 4. As disclosed herein,molecular profiling is performed to determine whether a gene or geneproduct is differentially expressed in a sample as compared to acontrol. The expression status of the gene or gene product is used toselect agents that are predicted to be efficacious or not. For example,Table 4 shows that overexpression of the ADA gene or protein points topentostatin as a possible treatment. On the other hand, underexpressionof the ADA gene or protein implicates resistance to cytarabine,suggesting that cytarabine is not an optimal treatment.

TABLE 4 Molecular Profiling Results and Predicted Treatments Gene NameExpression Status Candidate Agent(s) Possible Resistance ADAOverexpressed pentostatin ADA Underexpressed cytarabine AR Overexpressedabarelix, bicalutamide, flutamide, gonadorelin, goserelin, leuprolideASNS Underexpressed asparaginase, pegaspargase BCRP (ABCG2)Overexpressed cisplatin, carboplatin, irinotecan, topotecan BRCA1Underexpressed mitomycin BRCA2 Underexpressed mitomycin CD52Overexpressed alemtuzumab CDA Overexpressed cytarabine CES2Overexpressed irinotecan c-kit Overexpressed sorafenib, sunitinib,imatinib COX-2 Overexpressed celecoxib DCK Overexpressed gemcitabinecytarabine DHFR Underexpressed methotrexate, pemetrexed DHFROverexpressed methotrexate DNMT1 Overexpressed azacitidine, decitabineDNMT3A Overexpressed azacitidine, decitabine DNMT3B Overexpressedazacitidine, decitabine EGFR Overexpressed erlotinib, gefitinib,cetuximab, panitumumab EML4-ALK Overexpressed (present) crizotinib EPHA2Overexpressed dasatinib ER Overexpressed anastrazole, exemestane,fulvestrant, letrozole, megestrol, tamoxifen, medroxyprogesterone,toremifene, aminoglutethimide ERCC1 Overexpressed carboplatin, cisplatinGART Underexpressed pemetrexed HER-2 (ERBB2) Overexpressed trastuzumab,lapatinib HIF-1α Overexpressed sorafenib, sunitinib, bevacizumab IκB-αOverexpressed bortezomib MGMT Underexpressed temozolomide MGMTOverexpressed temozolomide MRP1 (ABCC1) Overexpressed etoposide,paclitaxel, docetaxel, vinblastine, vinorelbine, topotecan, teniposideP-gp (ABCB1) Overexpressed doxorubicin, etoposide, epirubicin,paclitaxel, docetaxel, vinblastine, vinorelbine, topotecan, teniposide,liposomal doxorubicin PDGFR-α Overexpressed sorafenib, sunitinib,imatinib PDGFR-β Overexpressed sorafenib, sunitinib, imatinib PROverexpressed exemestane, fulvestrant, gonadorelin, goserelin,medroxyprogesterone, megestrol, tamoxifen, toremifene RARA OverexpressedATRA RRM1 Underexpressed gemcitabine, hydroxyurea RRM2 Underexpressedgemcitabine, hydroxyurea RRM2B Underexpressed gemcitabine, hydroxyureaRXR-α Overexpressed bexarotene RXR-β Overexpressed bexarotene SPARCOverexpressed nab-paclitaxel SRC Overexpressed dasatinib SSTR2Overexpressed octreotide SSTR5 Overexpressed octreotide TOPO IOverexpressed irinotecan, topotecan TOPO IIα Overexpressed doxorubicin,epirubicin, liposomal-doxorubicin TOPO IIβ Overexpressed doxorubicin,epirubicin, liposomal-doxorubicin TS Underexpressed capecitabine, 5-fluorouracil, pemetrexed TS Overexpressed capecitabine, 5- fluorouracilVDR Overexpressed calcitriol, cholecalciferol VEGFR1 (Flt1)Overexpressed sorafenib, sunitinib, bevacizumab VEGFR2 Overexpressedsorafenib, sunitinib, bevacizumab VHL Underexpressed sorafenib,sunitinib

Table 5 presents a selection of illustrative rules for treatmentselection. The table is ordered by groups of related therapeutic agents.Each row describes a rule that maps the information derived frommolecular profiling with an indication of benefit or lack of benefit forthe therapeutic agent. Thus, the database contains a mapping oftreatments whose biological activity is known against cancer cells thathave alterations in certain genes or gene products, including gene copyalterations, chromosomal abnormalities, overexpression of orunderexpression of one or more genes or gene products, or have variousmutations. For each agent, a Lineage is presented as applicable whichcorresponds to a type of cancer associated with use of the agent. Agentswith Benefit are listed along with a Benefit Summary Statement thatdescribes molecular profiling information that relates to the predictedbeneficial agent. Similarly, agents with Lack of Benefit are listedalong with a Lack of Benefit Summary Statement that describes molecularprofiling information that relates to the lack of benefit associatedwith the agent. Finally, the molecular profiling Criteria are shown. Inthe criteria, results from analysis using DNA microarray (DMA), IHC,FISH, and mutation analysis (MA) for one or more biomarkers is listed.For microarray analysis, expression can be reported as over(overexpressed) or under (underexpressed). When these criteria are metaccording to the application of the molecular profiling techniques to asample, then the therapeutic agent or agents are predicted to have abenefit or lack of benefit as indicated in the corresponding row.

Further drug associations and rules that can be used in embodiments ofthe invention are found in U.S. Patent Application Publication20100304989, filed Feb. 12, 2010; International PCT Patent ApplicationWO/2010/093465, filed Feb. 11, 2010; and International PCT PatentApplication WO/2011/056688, filed Oct. 27, 2010; all of whichapplications are incorporated by reference herein in their entirety. Seee.g., “Table 4: Rules Summary for Treatment Selection” ofWO/2011/056688.

Lengthy table referenced here US20150024952A1-20150122-T00001 Pleaserefer to the end of the specification for access instructions.

The efficacy of various therapeutic agents given particular assayresults, such as those in Table 5 above, is derived from reviewing,analyzing and rendering conclusions on empirical evidence, such as thatis available the medical literature or other medical knowledge base. Theresults are used to guide the selection of certain therapeutic agents ina prioritized list for use in treatment of an individual. When molecularprofiling results are obtained, e.g., differential expression ormutation of a gene or gene product, the results can be compared againstthe database to guide treatment selection. The set of rules in thedatabase can be updated as new treatments and new treatment data becomeavailable. In some embodiments, the rules database is updatedcontinuously. In some embodiments, the rules database is updated on aperiodic basis. Any relevant correlative or comparative approach can beused to compare the molecular profiling results to the rules database.In one embodiment, a gene or gene product is identified asdifferentially expressed by molecular profiling. The rules database isqueried to select entries for that gene or gene product. Treatmentselection information selected from the rules database is extracted andused to select a treatment. The information, e.g., to recommend or notrecommend a particular treatment, can be dependent on whether the geneor gene product is over or underexpressed, or has other abnormalities atthe genetic or protein levels as compared to a reference. In some cases,multiple rules and treatments may be pulled from a database comprisingthe comprehensive rules set depending on the results of the molecularprofiling. In some embodiments, the treatment options are presented in aprioritized list. In some embodiments, the treatment options arepresented without prioritization information. In either case, anindividual, e.g., the treating physician or similar caregiver may choosefrom the available options.

The methods described herein are used to prolong survival of a subjectby providing personalized treatment. In some embodiments, the subjecthas been previously treated with one or more therapeutic agents to treatthe disease, e.g., a cancer. The cancer may be refractory to one ofthese agents, e.g., by acquiring drug resistance mutations. In someembodiments, the cancer is metastatic. In some embodiments, the subjecthas not previously been treated with one or more therapeutic agentsidentified by the method. Using molecular profiling, candidatetreatments can be selected regardless of the stage, anatomical location,or anatomical origin of the cancer cells.

Progression-free survival (PFS) denotes the chances of staying free ofdisease progression for an individual or a group of individualssuffering from a disease, e.g., a cancer, after initiating a course oftreatment. It can refer to the percentage of individuals in a groupwhose disease is likely to remain stable (e.g., not show signs ofprogression) after a specified duration of time. Progression-freesurvival rates are an indication of the effectiveness of a particulartreatment. Similarly, disease-free survival (DFS) denotes the chances ofstaying free of disease after initiating a particular treatment for anindividual or a group of individuals suffering from a cancer. It canrefer to the percentage of individuals in a group who are likely to befree of disease after a specified duration of time. Disease-freesurvival rates are an indication of the effectiveness of a particulartreatment. Treatment strategies can be compared on the basis of the PFSor DFS that is achieved in similar groups of patients. Disease-freesurvival is often used with the term overall survival when cancersurvival is described.

The candidate treatment selected by molecular profiling according to theinvention can be compared to a non-molecular profiling selectedtreatment by comparing the progression free survival (PFS) using therapyselected by molecular profiling (period B) with PFS for the most recenttherapy on which the patient has just progressed (period A). See FIG.32. In one setting, a PFS(B)/PFS(A) ratio ≧1.3 was used to indicate thatthe molecular profiling selected therapy provides benefit for patient(Robert Temple, Clinical measurement in drug evaluation. Edited by WuNingano and G. T. Thicker John Wiley and Sons Ltd. 1995; Von Hoff D. D.Clin Can Res. 4: 1079, 1999: Dhani et al. Clin Cancer Res. 15: 118-123,2009). Other methods of comparing the treatment selected by molecularprofiling to a non-molecular profiling selected treatment includedetermining response rate (RECIST) and percent of patients withoutprogression or death at 4 months. The term “about” as used in thecontext of a numerical value for PFS means a variation of +/− tenpercent (10%) relative to the numerical value. The PFS from a treatmentselected by molecular profiling can be extended by at least 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% as compared to anon-molecular profiling selected treatment. In some embodiments, the PFSfrom a treatment selected by molecular profiling can be extended by atleast 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or atleast about 1000% as compared to a non-molecular profiling selectedtreatment. In yet other embodiments, the PFS ratio (PFS on molecularprofiling selected therapy or new treatment/PFS on prior therapy ortreatment) is at least about 1.3. In yet other embodiments, the PFSratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0. In yet other embodiments, the PFS ratio is at least about 3, 4, 5,6, 7, 8, 9 or 10.

Similarly, the DFS can be compared in patients whose treatment isselected with or without molecular profiling. In embodiments, DFS from atreatment selected by molecular profiling is extended by at least 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% as compared to anon-molecular profiling selected treatment. In some embodiments, the DFSfrom a treatment selected by molecular profiling can be extended by atleast 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or atleast about 1000% as compared to a non-molecular profiling selectedtreatment. In yet other embodiments, the DFS ratio (DFS on molecularprofiling selected therapy or new treatment/DFS on prior therapy ortreatment) is at least about 1.3. In yet other embodiments, the DFSratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0. In yet other embodiments, the DFS ratio is at least about 3, 4, 5,6, 7, 8, 9 or 10.

In some embodiments, the candidate treatment of the invention will notincrease the PFS ratio or the DFS ratio in the patient, neverthelessmolecular profiling provides invaluable patient benefit. For example, insome instances no preferable treatment has been identified for thepatient. In such cases, molecular profiling provides a method toidentify a candidate treatment where none is currently identified. Themolecular profiling may extend PFS, DFS or lifespan by at least 1 week,2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks,2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 monthsor 2 years. The molecular profiling may extend PFS, DFS or lifespan byat least 2 V2 years, 3 years, 4 years, 5 years, or more. In someembodiments, the methods of the invention improve outcome so thatpatient is in remission.

The effectiveness of a treatment can be monitored by other measures. Acomplete response (CR) comprises a complete disappearance of thedisease: no disease is evident on examination, scans or other tests. Apartial response (PR) refers to some disease remaining in the body, butthere has been a decrease in size or number of the lesions by 30% ormore. Stable disease (SD) refers to a disease that has remainedrelatively unchanged in size and number of lesions. Generally, less thana 50% decrease or a slight increase in size would be described as stabledisease. Progressive disease (PD) means that the disease has increasedin size or number on treatment. In some embodiments, molecular profilingaccording to the invention results in a complete response or partialresponse. In some embodiments, the methods of the invention result instable disease. In some embodiments, the invention is able to achievestable disease where non-molecular profiling results in progressivedisease.

Computer Systems

Conventional data networking, application development and otherfunctional aspects of the systems (and components of the individualoperating components of the systems) may not be described in detailherein but are part of the invention. Furthermore, the connecting linesshown in the various figures contained herein are intended to representillustrative functional relationships and/or physical couplings betweenthe various elements. It should be noted that many alternative oradditional functional relationships or physical connections may bepresent in a practical system.

The various system components discussed herein may include one or moreof the following: a host server or other computing systems including aprocessor for processing digital data; a memory coupled to the processorfor storing digital data; an input digitizer coupled to the processorfor inputting digital data; an application program stored in the memoryand accessible by the processor for directing processing of digital databy the processor; a display device coupled to the processor and memoryfor displaying information derived from digital data processed by theprocessor; and a plurality of databases. Various databases used hereinmay include: patient data such as family history, demography andenvironmental data, biological sample data, prior treatment and protocoldata, patient clinical data, molecular profiling data of biologicalsamples, data on therapeutic drug agents and/or investigative drugs, agene library, a disease library, a drug library, patient tracking data,file management data, financial management data, billing data and/orlike data useful in the operation of the system. As those skilled in theart will appreciate, user computer may include an operating system(e.g., Windows NT, 95/98/2000, OS2, UNIX, Linux, Solaris, MacOS, etc.)as well as various conventional support software and drivers typicallyassociated with computers. The computer may include any suitablepersonal computer, network computer, workstation, minicomputer,mainframe or the like. User computer can be in a home ormedical/business environment with access to a network. In anillustrative embodiment, access is through a network or the Internetthrough a commercially-available web-browser software package.

As used herein, the term “network” shall include any electroniccommunications means which incorporates both hardware and softwarecomponents of such. Communication among the parties may be accomplishedthrough any suitable communication channels, such as, for example, atelephone network, an extranet, an intranet, Internet, point ofinteraction device, personal digital assistant (e.g., Palm Pilot®,Blackberry®), cellular phone, kiosk, etc.), online communications,satellite communications, off-line communications, wirelesscommunications, transponder communications, local area network (LAN),wide area network (WAN), networked or linked devices, keyboard, mouseand/or any suitable communication or data input modality. Moreover,although the system is frequently described herein as being implementedwith TCP/IP communications protocols, the system may also be implementedusing IPX, Appletalk, IP-6, NetBIOS, OSI or any number of existing orfuture protocols. If the network is in the nature of a public network,such as the Internet, it may be advantageous to presume the network tobe insecure and open to eavesdroppers. Specific information related tothe protocols, standards, and application software used in connectionwith the Internet is generally known to those skilled in the art and, assuch, need not be detailed herein. See, for example, DILIP NAIK,INTERNET STANDARDS AND PROTOCOLS (1998); JAVA 2 COMPLETE, variousauthors, (Sybex 1999); DEBORAH RAY AND ERIC RAY, MASTERING HTML 4.0(1997); and LOSHIN, TCP/IP CLEARLY EXPLAINED (1997) and DAVID GOURLEYAND BRIAN TOTTY, HTTP, THE DEFINITIVE GUIDE (2002), the contents ofwhich are hereby incorporated by reference.

The various system components may be independently, separately orcollectively suitably coupled to the network via data links whichincludes, for example, a connection to an Internet Service Provider(ISP) over the local loop as is typically used in connection withstandard modem communication, cable modem, Dish networks, ISDN, DigitalSubscriber Line (DSL), or various wireless communication methods, see,e.g., GILBERT HELD, UNDERSTANDING DATA COMMUNICATIONS (1996), which ishereby incorporated by reference. It is noted that the network may beimplemented as other types of networks, such as an interactivetelevision (ITV) network. Moreover, the system contemplates the use,sale or distribution of any goods, services or information over anynetwork having similar functionality described herein.

As used herein, “transmit” may include sending electronic data from onesystem component to another over a network connection. Additionally, asused herein, “data” may include encompassing information such ascommands, queries, files, data for storage, and the like in digital orany other form.

The system contemplates uses in association with web services, utilitycomputing, pervasive and individualized computing, security and identitysolutions, autonomic computing, commodity computing, mobility andwireless solutions, open source, biometrics, grid computing and/or meshcomputing.

Any databases discussed herein may include relational, hierarchical,graphical, or object-oriented structure and/or any other databaseconfigurations. Common database products that may be used to implementthe databases include DB2 by IBM (White Plains, N.Y.), various databaseproducts available from Oracle Corporation (Redwood Shores, Calif.),Microsoft Access or Microsoft SQL Server by Microsoft Corporation(Redmond, Wash.), or any other suitable database product. Moreover, thedatabases may be organized in any suitable manner, for example, as datatables or lookup tables. Each record may be a single file, a series offiles, a linked series of data fields or any other data structure.Association of certain data may be accomplished through any desired dataassociation technique such as those known or practiced in the art. Forexample, the association may be accomplished either manually orautomatically. Automatic association techniques may include, forexample, a database search, a database merge, GREP, AGREP, SQL, using akey field in the tables to speed searches, sequential searches throughall the tables and files, sorting records in the file according to aknown order to simplify lookup, and/or the like. The association stepmay be accomplished by a database merge function, for example, using a“key field” in pre-selected databases or data sectors.

More particularly, a “key field” partitions the database according tothe high-level class of objects defined by the key field. For example,certain types of data may be designated as a key field in a plurality ofrelated data tables and the data tables may then be linked on the basisof the type of data in the key field. The data corresponding to the keyfield in each of the linked data tables is preferably the same or of thesame type. However, data tables having similar, though not identical,data in the key fields may also be linked by using AGREP, for example.In accordance with one embodiment, any suitable data storage techniquemay be used to store data without a standard format. Data sets may bestored using any suitable technique, including, for example, storingindividual files using an ISO/IEC 7816-4 file structure; implementing adomain whereby a dedicated file is selected that exposes one or moreelementary files containing one or more data sets; using data setsstored in individual files using a hierarchical filing system; data setsstored as records in a single file (including compression, SQLaccessible, hashed via one or more keys, numeric, alphabetical by firsttuple, etc.); Binary Large Object (BLOB); stored as ungrouped dataelements encoded using ISO/IEC 7816-6 data elements; stored as ungroupeddata elements encoded using ISO/IEC Abstract Syntax Notation (ASN.1) asin ISO/IEC 8824 and 8825; and/or other proprietary techniques that mayinclude fractal compression methods, image compression methods, etc.

In one illustrative embodiment, the ability to store a wide variety ofinformation in different formats is facilitated by storing theinformation as a BLOB. Thus, any binary information can be stored in astorage space associated with a data set. The BLOB method may store datasets as ungrouped data elements formatted as a block of binary via afixed memory offset using either fixed storage allocation, circularqueue techniques, or best practices with respect to memory management(e.g., paged memory, least recently used, etc.). By using BLOB methods,the ability to store various data sets that have different formatsfacilitates the storage of data by multiple and unrelated owners of thedata sets. For example, a first data set which may be stored may beprovided by a first party, a second data set which may be stored may beprovided by an unrelated second party, and yet a third data set whichmay be stored, may be provided by a third party unrelated to the firstand second party. Each of these three illustrative data sets may containdifferent information that is stored using different data storageformats and/or techniques. Further, each data set may contain subsets ofdata that also may be distinct from other subsets.

As stated above, in various embodiments, the data can be stored withoutregard to a common format. However, in one illustrative embodiment, thedata set (e.g., BLOB) may be annotated in a standard manner whenprovided for manipulating the data. The annotation may comprise a shortheader, trailer, or other appropriate indicator related to each data setthat is configured to convey information useful in managing the variousdata sets. For example, the annotation may be called a “conditionheader”, “header”, “trailer”, or “status”, herein, and may comprise anindication of the status of the data set or may include an identifiercorrelated to a specific issuer or owner of the data. Subsequent bytesof data may be used to indicate for example, the identity of the issueror owner of the data, user, transaction/membership account identifier orthe like. Each of these condition annotations are further discussedherein.

The data set annotation may also be used for other types of statusinformation as well as various other purposes. For example, the data setannotation may include security information establishing access levels.The access levels may, for example, be configured to permit only certainindividuals, levels of employees, companies, or other entities to accessdata sets, or to permit access to specific data sets based on thetransaction, issuer or owner of data, user or the like. Furthermore, thesecurity information may restrict/permit only certain actions such asaccessing, modifying, and/or deleting data sets. In one example, thedata set annotation indicates that only the data set owner or the userare permitted to delete a data set, various identified users may bepermitted to access the data set for reading, and others are altogetherexcluded from accessing the data set. However, other access restrictionparameters may also be used allowing various entities to access a dataset with various permission levels as appropriate. The data, includingthe header or trailer may be received by a standalone interaction deviceconfigured to add, delete, modify, or augment the data in accordancewith the header or trailer.

One skilled in the art will also appreciate that, for security reasons,any databases, systems, devices, servers or other components of thesystem may consist of any combination thereof at a single location or atmultiple locations, wherein each database or system includes any ofvarious suitable security features, such as firewalls, access codes,encryption, decryption, compression, decompression, and/or the like.

The computing unit of the web client may be further equipped with anInternet browser connected to the Internet or an intranet using standarddial-up, cable, DSL or any other Internet protocol known in the art.Transactions originating at a web client may pass through a firewall inorder to prevent unauthorized access from users of other networks.Further, additional firewalls may be deployed between the varyingcomponents of CMS to further enhance security.

Firewall may include any hardware and/or software suitably configured toprotect CMS components and/or enterprise computing resources from usersof other networks. Further, a firewall may be configured to limit orrestrict access to various systems and components behind the firewallfor web clients connecting through a web server. Firewall may reside invarying configurations including Stateful Inspection, Proxy based andPacket Filtering among others. Firewall may be integrated within an webserver or any other CMS components or may further reside as a separateentity.

The computers discussed herein may provide a suitable website or otherInternet-based graphical user interface which is accessible by users. Inone embodiment, the Microsoft Internet Information Server (IIS),Microsoft Transaction Server (MTS), and Microsoft SQL Server, are usedin conjunction with the Microsoft operating system, Microsoft NT webserver software, a Microsoft SQL Server database system, and a MicrosoftCommerce Server. Additionally, components such as Access or MicrosoftSQL Server, Oracle, Sybase, Informix MySQL, Interbase, etc., may be usedto provide an Active Data Object (ADO) compliant database managementsystem.

Any of the communications, inputs, storage, databases or displaysdiscussed herein may be facilitated through a website having web pages.The term “web page” as it is used herein is not meant to limit the typeof documents and applications that might be used to interact with theuser. For example, a typical website might include, in addition tostandard HTML documents, various forms, Java applets, JavaScript, activeserver pages (ASP), common gateway interface scripts (CGI), extensiblemarkup language (XML), dynamic HTML, cascading style sheets (CSS),helper applications, plug-ins, and the like. A server may include a webservice that receives a request from a web server, the request includinga URL (http://yahoo.com/stockquotes/ge) and an IP address(123.56.789.234). The web server retrieves the appropriate web pages andsends the data or applications for the web pages to the IP address. Webservices are applications that are capable of interacting with otherapplications over a communications means, such as the internet. Webservices are typically based on standards or protocols such as XML,XSLT, SOAP, WSDL and UDDI. Web services methods are well known in theart, and are covered in many standard texts. See, e.g., ALEX NGHLEM, ITWEB SERVICES: A ROADMAP FOR THE ENTERPRISE (2003), hereby incorporatedby reference.

The web-based clinical database for the system and method of the presentinvention preferably has the ability to upload and store clinical datafiles in native formats and is searchable on any clinical parameter. Thedatabase is also scalable and may use an EAV data model (metadata) toenter clinical annotations from any study for easy integration withother studies. In addition, the web-based clinical database is flexibleand may be XML and XSLT enabled to be able to add user customizedquestions dynamically. Further, the database includes exportability toCDISC ODM.

Practitioners will also appreciate that there are a number of methodsfor displaying data within a browser-based document. Data may berepresented as standard text or within a fixed list, scrollable list,drop-down list, editable text field, fixed text field, pop-up window,and the like. Likewise, there are a number of methods available formodifying data in a web page such as, for example, free text entry usinga keyboard, selection of menu items, check boxes, option boxes, and thelike.

The system and method may be described herein in terms of functionalblock components, screen shots, optional selections and variousprocessing steps. It should be appreciated that such functional blocksmay be realized by any number of hardware and/or software componentsconfigured to perform the specified functions. For example, the systemmay employ various integrated circuit components, e.g., memory elements,processing elements, logic elements, look-up tables, and the like, whichmay carry out a variety of functions under the control of one or moremicroprocessors or other control devices. Similarly, the softwareelements of the system may be implemented with any programming orscripting language such as C, C-HE, Macromedia Cold Fusion, MicrosoftActive Server Pages, Java, COBOL, assembler, PEAL, Visual Basic, SQLStored Procedures, extensible markup language (XML), with the variousalgorithms being implemented with any combination of data structures,objects, processes, routines or other programming elements. Further, itshould be noted that the system may employ any number of conventionaltechniques for data transmission, signaling, data processing, networkcontrol, and the like. Still further, the system could be used to detector prevent security issues with a client-side scripting language, suchas JavaScript, VBScript or the like. For a basic introduction ofcryptography and network security, see any of the following references:(1) “Applied Cryptography: Protocols, Algorithms, And Source Code In C,”by Bruce Schneier, published by John Wiley & Sons (second edition,1995); (2) “Java Cryptography” by Jonathan Knudson, published byO'Reilly & Associates (1998); (3) “Cryptography & Network Security:Principles & Practice” by William Stallings, published by Prentice Hall;all of which are hereby incorporated by reference.

As used herein, the term “end user”, “consumer”, “customer”, “client”,“treating physician”, “hospital”, or “business” may be usedinterchangeably with each other, and each shall mean any person, entity,machine, hardware, software or business. Each participant is equippedwith a computing device in order to interact with the system andfacilitate online data access and data input. The customer has acomputing unit in the form of a personal computer, although other typesof computing units may be used including laptops, notebooks, hand heldcomputers, set-top boxes, cellular telephones, touch-tone telephones andthe like. The owner/operator of the system and method of the presentinvention has a computing unit implemented in the form of acomputer-server, although other implementations are contemplated by thesystem including a computing center shown as a main frame computer, amini-computer, a PC server, a network of computers located in the sameof different geographic locations, or the like. Moreover, the systemcontemplates the use, sale or distribution of any goods, services orinformation over any network having similar functionality describedherein.

In one illustrative embodiment, each client customer may be issued an“account” or “account number”. As used herein, the account or accountnumber may include any device, code, number, letter, symbol, digitalcertificate, smart chip, digital signal, analog signal, biometric orother identifier/indicia suitably configured to allow the consumer toaccess, interact with or communicate with the system (e.g., one or moreof an authorization/access code, personal identification number (PIN),Internet code, other identification code, and/or the like). The accountnumber may optionally be located on or associated with a charge card,credit card, debit card, prepaid card, embossed card, smart card,magnetic stripe card, bar code card, transponder, radio frequency cardor an associated account. The system may include or interface with anyof the foregoing cards or devices, or a fob having a transponder andRFID reader in RF communication with the fob. Although the system mayinclude a fob embodiment, the invention is not to be so limited. Indeed,system may include any device having a transponder which is configuredto communicate with RFID reader via RF communication. Typical devicesmay include, for example, a key ring, tag, card, cell phone, wristwatchor any such form capable of being presented for interrogation. Moreover,the system, computing unit or device discussed herein may include a“pervasive computing device,” which may include a traditionallynon-computerized device that is embedded with a computing unit. Theaccount number may be distributed and stored in any form of plastic,electronic, magnetic, radio frequency, wireless, audio and/or opticaldevice capable of transmitting or downloading data from itself to asecond device.

As will be appreciated by one of ordinary skill in the art, the systemmay be embodied as a customization of an existing system, an add-onproduct, upgraded software, a standalone system, a distributed system, amethod, a data processing system, a device for data processing, and/or acomputer program product. Accordingly, the system may take the form ofan entirely software embodiment, an entirely hardware embodiment, or anembodiment combining aspects of both software and hardware. Furthermore,the system may take the form of a computer program product on acomputer-readable storage medium having computer-readable program codemeans embodied in the storage medium. Any suitable computer-readablestorage medium may be used, including hard disks, CD-ROM, opticalstorage devices, magnetic storage devices, and/or the like.

The system and method is described herein with reference to screenshots, block diagrams and flowchart illustrations of methods, apparatus(e.g., systems), and computer program products according to variousembodiments. It will be understood that each functional block of theblock diagrams and the flowchart illustrations, and combinations offunctional blocks in the block diagrams and flowchart illustrations,respectively, can be implemented by computer program instructions.

These computer program instructions may be loaded onto a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructionsthat execute on the computer or other programmable data processingapparatus create means for implementing the functions specified in theflowchart block or blocks. These computer program instructions may alsobe stored in a computer-readable memory that can direct a computer orother programmable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instruction meanswhich implement the function specified in the flowchart block or blocks.The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer-implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing the functions specified in theflowchart block or blocks.

Accordingly, functional blocks of the block diagrams and flowchartillustrations support combinations of means for performing the specifiedfunctions, combinations of steps for performing the specified functions,and program instruction means for performing the specified functions. Itwill also be understood that each functional block of the block diagramsand flowchart illustrations, and combinations of functional blocks inthe block diagrams and flowchart illustrations, can be implemented byeither special purpose hardware-based computer systems which perform thespecified functions or steps, or suitable combinations of specialpurpose hardware and computer instructions. Further, illustrations ofthe process flows and the descriptions thereof may make reference touser windows, web pages, websites, web forms, prompts, etc.Practitioners will appreciate that the illustrated steps describedherein may comprise in any number of configurations including the use ofwindows, web pages, web forms, popup windows, prompts and the like. Itshould be further appreciated that the multiple steps as illustrated anddescribed may be combined into single web pages and/or windows but havebeen expanded for the sake of simplicity. In other cases, stepsillustrated and described as single process steps may be separated intomultiple web pages and/or windows but have been combined for simplicity.

Molecular Profiling Methods

FIG. 1 illustrates a block diagram of an illustrative embodiment of asystem 10 for determining individualized medical intervention for aparticular disease state that uses molecular profiling of a patient'sbiological specimen. System 10 includes a user interface 12, a hostserver 14 including a processor 16 for processing data, a memory 18coupled to the processor, an application program 20 stored in the memory18 and accessible by the processor 16 for directing processing of thedata by the processor 16, a plurality of internal databases 22 andexternal databases 24, and an interface with a wired or wirelesscommunications network 26 (such as the Internet, for example). System 10may also include an input digitizer 28 coupled to the processor 16 forinputting digital data from data that is received from user interface12.

User interface 12 includes an input device 30 and a display 32 forinputting data into system 10 and for displaying information derivedfrom the data processed by processor 16. User interface 12 may alsoinclude a printer 34 for printing the information derived from the dataprocessed by the processor 16 such as patient reports that may includetest results for targets and proposed drug therapies based on the testresults.

Internal databases 22 may include, but are not limited to, patientbiological sample/specimen information and tracking, clinical data,patient data, patient tracking, file management, study protocols,patient test results from molecular profiling, and billing informationand tracking. External databases 24 may include, but are not limited to,drug libraries, gene libraries, disease libraries, and public andprivate databases such as UniGene, OMIM, GO, TIGR, GenBank, KEGG andBiocarta.

Various methods may be used in accordance with system 10. FIG. 2 shows aflowchart of an illustrative embodiment of a method 50 for determiningindividualized medical intervention for a particular disease state thatuses molecular profiling of a patient's biological specimen that is nondisease specific. In order to determine a medical intervention for aparticular disease state using molecular profiling that is independentof disease lineage diagnosis (i.e. not single disease restricted), atleast one test is performed for at least one target from a biologicalsample of a diseased patient in step 52. A target is defined as anymolecular finding that may be obtained from molecular testing. Forexample, a target may include one or more genes, one or more geneexpressed proteins, one or more molecular mechanisms, and/orcombinations of such. For example, the expression level of a target canbe determined by the analysis of mRNA levels or the target or gene, orprotein levels of the gene. Tests for finding such targets may include,but are not limited, fluorescent in-situ hybridization (FISH), anin-situ hybridization (ISH), and other molecular tests known to thoseskilled in the art. PCR-based methods, such as real-time PCR orquantitative PCR can be used. Furthermore, microarray analysis, such asa comparative genomic hybridization (CGH) micro array, a singlenucleotide polymorphism (SNP) microarray, a proteomic array, or antibodyarray analysis can also be used in the methods disclosed herein. In someembodiments, microarray analysis comprises identifying whether a gene isup-regulated or down-regulated relative to a reference with asignificance of p<0.001. Tests or analyses of targets can also compriseimmunohistochemical (IHC) analysis. In some embodiments, INC analysiscomprises determining whether 30% or more of a sample is stained, if thestaining intensity is +2 or greater, or both.

Furthermore, the methods disclosed herein also including profiling morethan one target. For example, the expression of a plurality of genes canbe identified. Furthermore, identification of a plurality of targets ina sample can be by one method or by various means. For example, theexpression of a first gene can be determined by one method and theexpression level of a second gene determined by a different method.Alternatively, the same method can be used to detect the expressionlevel of the first and second gene. For example, the first method can beINC and the second by microarray analysis, such as detecting the geneexpression of a gene.

In some embodiments, molecular profiling can also including identifyinga genetic variant, such as a mutation, polymorphism (such as a SNP),deletion, or insertion of a target. For example, identifying a SNP in agene can be determined by microarray analysis, real-time PCR, orsequencing. Other methods disclosed herein can also be used to identifyvariants of one or more targets.

Accordingly, one or more of the following may be performed: an IHCanalysis in step 54, a microanalysis in step 56, and other moleculartests know to those skilled in the art in step 58.

Biological samples are obtained from diseased patients by taking abiopsy of a tumor, conducting minimally invasive surgery if no recenttumor is available, obtaining a sample of the patient's blood, or asample of any other biological fluid including, but not limited to, cellextracts, nuclear extracts, cell lysates or biological products orsubstances of biological origin such as excretions, blood, sera, plasma,urine, sputum, tears, feces, saliva, membrane extracts, and the like.

In step 60, a determination is made as to whether one or more of thetargets that were tested for in step 52 exhibit a change in expressioncompared to a normal reference for that particular target. In oneillustrative method of the invention, an INC analysis may be performedin step 54 and a determination as to whether any targets from the IHCanalysis exhibit a change in expression is made in step 64 bydetermining whether 30% or more of the biological sample cells were +2or greater staining for the particular target. It will be understood bythose skilled in the art that there will be instances where +1 orgreater staining will indicate a change in expression in that stainingresults may vary depending on the technician performing the test andtype of target being tested. In another illustrative embodiment of theinvention, a micro array analysis may be performed in step 56 and adetermination as to whether any targets from the micro array analysisexhibit a change in expression is made in step 66 by identifying whichtargets are up-regulated or down-regulated by determining whether thefold change in expression for a particular target relative to a normaltissue of origin reference is significant at p<0.001. A change inexpression may also be evidenced by an absence of one or more genes,gene expressed proteins, molecular mechanisms, or other molecularfindings.

After determining which targets exhibit a change in expression in step60, at least one non-disease specific agent is identified that interactswith each target having a changed expression in step 70. An agent may beany drug or compound having a therapeutic effect. A non-disease specificagent is a therapeutic drug or compound not previously associated withtreating the patient's diagnosed disease that is capable of interactingwith the target from the patient's biological sample that has exhibiteda change in expression. Some of the non-disease specific agents thathave been found to interact with specific targets found in differentcancer patients are shown in Table 6 below.

TABLE 6 Illustrative target-drug associations Patients Target(s) FoundTreatment(s) Advanced Pancreatic HER 2/neu (IHC/Array) Herceptin ™Cancer Advanced Pancreatic EGFR (IHC), HIF 1α Erbitux ™, CancerRapamycin ™ Advanced Ovarian Cancer ERCC3 (Array) Irofulvene AdvancedAdenoid Cystic Vitamin D receptors, Calcitriol ™ Carcinoma Androgenreceptors Flutamide ™

Finally, in step 80, a patient profile report may be provided whichincludes the patient's test results for various targets and any proposedtherapies based on those results. An illustrative patient profile report100 is shown in FIGS. 3A-3D. Patient profile report 100 shown in FIG. 3Aidentifies the targets tested 102, those targets tested that exhibitedsignificant changes in expression 104, and proposed non-disease specificagents for interacting with the targets 106. Patient profile report 100shown in FIG. 3B identifies the results 108 of immunohistochemicalanalysis for certain gene expressed proteins 110 and whether a geneexpressed protein is a molecular target 112 by determining whether 30%or more of the tumor cells were +2 or greater staining. Report 100 alsoidentifies immunohistochemical tests that were not performed 114.Patient profile report 100 shown in FIG. 3C identifies the genesanalyzed 116 with a micro array analysis and whether the genes wereunder expressed or over expressed 118 compared to a reference. Finally,patient profile report 100 shown in FIG. 3D identifies the clinicalhistory 120 of the patient and the specimens that were submitted 122from the patient. Molecular profiling techniques can be performedanywhere, e.g., a foreign country, and the results sent by network to anappropriate party, e.g., the patient, a physician, lab or other partylocated remotely.

FIG. 4 shows a flowchart of an illustrative embodiment of a method 200for identifying a drug therapy/agent capable of interacting with atarget. In step 202, a molecular target is identified which exhibits achange in expression in a number of diseased individuals. Next, in step204, a drug therapy/agent is administered to the diseased individuals.After drug therapy/agent administration, any changes in the moleculartarget identified in step 202 are identified in step 206 in order todetermine if the drug therapy/agent administered in step 204 interactswith the molecular targets identified in step 202. If it is determinedthat the drug therapy/agent administered in step 204 interacts with amolecular target identified in step 202, the drug therapy/agent may beapproved for treating patients exhibiting a change in expression of theidentified molecular target instead of approving the drug therapy/agentfor a particular disease.

FIGS. 5-14 are flowcharts and diagrams illustrating various parts of aninformation-based personalized medicine drug discovery system and methodin accordance with the present invention. FIG. 5 is a diagram showing anillustrative clinical decision support system of the information-basedpersonalized medicine drug discovery system and method of the presentinvention. Data obtained through clinical research and clinical caresuch as clinical trial data, biomedical/molecular imaging data,genomics/proteomics/chemical library/literature/expert curation,biospecimen tracking/LIMS, family history/environmental records, andclinical data are collected and stored as databases and datamarts withina data warehouse. FIG. 6 is a diagram showing the flow of informationthrough the clinical decision support system of the information-basedpersonalized medicine drug discovery system and method of the presentinvention using web services. A user interacts with the system byentering data into the system via form-based entry/upload of data sets,formulating queries and executing data analysis jobs, and acquiring andevaluating representations of output data. The data warehouse in the webbased system is where data is extracted, transformed, and loaded fromvarious database systems. The data warehouse is also where commonformats, mapping and transformation occurs. The web based system alsoincludes datamarts which are created based on data views of interest.

A flow chart of an illustrative clinical decision support system of theinformation-based personalized medicine drug discovery system and methodof the present invention is shown in FIG. 7. The clinical informationmanagement system includes the laboratory information management systemand the medical information contained in the data warehouses anddatabases includes medical information libraries, such as druglibraries, gene libraries, and disease libraries, in addition toliterature text mining. Both the information management systems relatingto particular patients and the medical information databases and datawarehouses come together at a data junction center where diagnosticinformation and therapeutic options can be obtained. A financialmanagement system may also be incorporated in the clinical decisionsupport system of the information-based personalized medicine drugdiscovery system and method of the present invention.

FIG. 8 is a diagram showing an illustrative biospecimen tracking andmanagement system which may be used as part of the information-basedpersonalized medicine drug discovery system and method of the presentinvention. FIG. 8 shows two host medical centers which forward specimensto a tissue/blood bank. The specimens may go through laboratory analysisprior to shipment. Research may also be conducted on the samples viamicro array, genotyping, and proteomic analysis. This information can beredistributed to the tissue/blood bank. FIG. 9 depicts a flow chart ofan illustrative biospecimen tracking and management system which may beused with the information-based personalized medicine drug discoverysystem and method of the present invention. The host medical centerobtains samples from patients and then ships the patient samples to amolecular profiling laboratory which may also perform RNA and DNAisolation and analysis.

A diagram showing a method for maintaining a clinical standardizedvocabulary for use with the information-based personalized medicine drugdiscovery system and method of the present invention is shown in FIG.10. FIG. 10 illustrates how physician observations and patientinformation associated with one physician's patient may be madeaccessible to another physician to enable the other physician to use thedata in making diagnostic and therapeutic decisions for their patients.

FIG. 11 shows a schematic of an illustrative microarray gene expressiondatabase which may be used as part of the information-based personalizedmedicine drug discovery system and method of the present invention. Themicro array gene expression database includes both external databasesand internal databases which can be accessed via the web based system.External databases may include, but are not limited to, UniGene, GO,TIGR, GenBank, KEGG. The internal databases may include, but are notlimited to, tissue tracking, LIMS, clinical data, and patient tracking.FIG. 12 shows a diagram of an illustrative micro array gene expressiondatabase data warehouse which may be used as part of theinformation-based personalized medicine drug discovery system and methodof the present invention. Laboratory data, clinical data, and patientdata may all be housed in the micro array gene expression database datawarehouse and the data may in turn be accessed by public/private releaseand used by data analysis tools.

Another schematic showing the flow of information through aninformation-based personalized medicine drug discovery system and methodof the present invention is shown in FIG. 13. Like FIG. 7, the schematicincludes clinical information management, medical and literatureinformation management, and financial management of theinformation-based personalized medicine drug discovery system and methodof the present invention. FIG. 14 is a schematic showing an illustrativenetwork of the information-based personalized medicine drug discoverysystem and method of the present invention. Patients, medicalpractitioners, host medical centers, and labs all share and exchange avariety of information in order to provide a patient with a proposedtherapy or agent based on various identified targets.

FIGS. 15-25 are computer screen print outs associated with various partsof the information-based personalized medicine drug discovery system andmethod shown in FIGS. 5-14. FIGS. 15 and 16 show computer screens wherephysician information and insurance company information is entered onbehalf of a client. FIGS. 17-19 show computer screens in whichinformation can be entered for ordering analysis and tests on patientsamples.

FIG. 20 is a computer screen showing micro array analysis results ofspecific genes tested with patient samples. This information andcomputer screen is similar to the information detailed in the patientprofile report shown in FIG. 3C. FIG. 22 is a computer screen that showsimmunohistochemistry test results for a particular patient for variousgenes. This information is similar to the information contained in thepatient profile report shown in FIG. 3B.

FIG. 21 is a computer screen showing selection options for findingparticular patients, ordering tests and/or results, issuing patientreports, and tracking current cases/patients.

FIG. 23 is a computer screen which outlines some of the steps forcreating a patient profile report as shown in FIGS. 3A through 3D. FIG.24 shows a computer screen for ordering an immunohistochemistry test ona patient sample and FIG. 25 shows a computer screen for enteringinformation regarding a primary tumor site for micro array analysis. Itwill be understood by those skilled in the art that any number andvariety of computer screens may be used to enter the informationnecessary for using the information-based personalized medicine drugdiscovery system and method of the present invention and to obtaininformation resulting from using the information-based personalizedmedicine drug discovery system and method of the present invention.

FIGS. 26-31 represent tables that show the frequency of a significantchange in expression of certain genes and/or gene expressed proteins bytumor type, i.e. the number of times that a gene and/or gene expressedprotein was flagged as a target by tumor type as being significantlyoverexpressed or underexpressed (see also Examples 1-3). The tables showthe total number of times a gene and/or gene expressed protein wasoverexpressed or underexpressed in a particular tumor type and whetherthe change in expression was determined by immunohistochemistry analysis(FIG. 26, FIG. 28) or microarray analysis (FIGS. 27, 30). The tablesalso identify the total number of times an overexpression of any geneexpressed protein occurred in a particular tumor type usingimmunohistochemistry and the total number of times an overexpression orunderexpression of any gene occurred in a particular tumor type usinggene microarray analysis.

Molecular Profiling Targets

The present invention provides methods and systems for analyzingdiseased tissue using molecular profiling as previously described above.Because the methods rely on analysis of the characteristics of the tumorunder analysis, the methods can be applied in for any tumor or any stageof disease, such an advanced stage of disease or a metastatic tumor ofunknown origin. As described herein, a tumor or cancer sample isanalyzed for molecular characteristics in order to predict or identify acandidate therapeutic treatment. The molecular characteristics caninclude the expression of genes or gene products, assessment of genecopy number, or mutational analysis. Any relevant determinablecharacteristic that can assist in prediction or identification of acandidate therapeutic can be included within the methods of theinvention.

The biomarker patterns or biomarker signature sets can be determined fortumor types, diseased tissue types, or diseased cells including withoutlimitation adipose, adrenal cortex, adrenal gland, adrenalgland—medulla, appendix, bladder, blood vessel, bone, bone cartilage,brain, breast, cartilage, cervix, colon, colon sigmoid, dendritic cells,skeletal muscle, endometrium, esophagus, fallopian tube, fibroblast,gallbladder, kidney, larynx, liver, lung, lymph node, melanocytes,mesothelial lining, myoepithelial cells, osteoblasts, ovary, pancreas,parotid, prostate, salivary gland, sinus tissue, skeletal muscle, skin,small intestine, smooth muscle, stomach, synovium, joint lining tissue,tendon, testis, thymus, thyroid, uterus, and uterus corpus.

The methods of the present invention can be used for selecting atreatment of any cancer or tumor type, including but not limited tobreast cancer (including HER2+ breast cancer, HER2− breast cancer,ER/PR+, HER2− breast cancer, or triple negative breast cancer),pancreatic cancer, cancer of the colon and/or rectum, leukemia, skincancer, bone cancer, prostate cancer, liver cancer, lung cancer, braincancer, cancer of the larynx, gallbladder, parathyroid, thyroid,adrenal, neural tissue, head and neck, stomach, bronchi, kidneys, basalcell carcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma,veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, islet cell carcinoma, primary brain tumor, acute and chroniclymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma,intestinal ganglioneuroma, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyoma,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoides, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, and epidermoid carcinomas. The cancer or tumor can comprise,without limitation, a carcinoma, a sarcoma, a lymphoma or leukemia, agerm cell tumor, a blastoma, or other cancers. Carcinomas that can beassessed using the subject methods include without limitation epithelialneoplasms, squamous cell neoplasms, squamous cell carcinoma, basal cellneoplasms basal cell carcinoma, transitional cell papillomas andcarcinomas, adenomas and adenocarcinomas (glands), adenoma,adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma,vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cysticcarcinoma, carcinoid tumor of appendix, prolactinoma, oncocytoma,hurthle cell adenoma, renal cell carcinoma, grawitz tumor, multipleendocrine adenomas, endometrioid adenoma, adnexal and skin appendageneoplasms, mucoepidermoid neoplasms, cystic, mucinous and serousneoplasms, cystadenoma, pseudomyxoma peritonei, ductal, lobular andmedullary neoplasms, acinar cell neoplasms, complex epithelialneoplasms, warthin's tumor, thymoma, specialized gonadal neoplasms, sexcord stromal tumor, thecoma, granulosa cell tumor, arrhenoblastoma,sertoli leydig cell tumor, glomus tumors, paraganglioma,pheochromocytoma, glomus tumor, nevi and melanomas, melanocytic nevus,malignant melanoma, melanoma, nodular melanoma, dysplastic nevus,lentigo maligna melanoma, superficial spreading melanoma, and malignantacral lentiginous melanoma. Sarcoma that can be assessed using thesubject methods include without limitation Askin's tumor, botryodies,chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma,malignant schwannoma, osteosarcoma, soft tissue sarcomas including:alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes,dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor,epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletalosteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma,kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma,rhabdomyosarcoma, and synovialsarcoma. Lymphoma and leukemia that can beassessed using the subject methods include without limitation chroniclymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma (such as waldenstrommacroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma,plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chaindiseases, extranodal marginal zone B cell lymphoma, also called maltlymphoma, nodal marginal zone B cell lymphoma (nmzl), follicularlymphoma, mantle cell lymphoma, diffuse large B cell lymphoma,mediastinal (thymic) large B cell lymphoma, intravascular large B celllymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cellprolymphocytic leukemia, T cell large granular lymphocytic leukemia,aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodalNK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma,hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosisfungoides/sezary syndrome, primary cutaneous CD30-positive T celllymphoproliferative disorders, primary cutaneous anaplastic large celllymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma,peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma,classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity,lymphocyte-rich, lymphocyte depleted or not depleted), and nodularlymphocyte-predominant Hodgkin lymphoma. Germ cell tumors that can beassessed using the subject methods include without limitation germinoma,dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonalcarcinoma, endodermal sinus turmor, choriocarcinoma, teratoma,polyembryoma, and gonadoblastoma. Blastoma includes without limitationnephroblastoma, medulloblastoma, and retinoblastoma. Other cancersinclude without limitation labial carcinoma, larynx carcinoma,hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma,gastric carcinoma, adenocarcinoma, thyroid cancer (medullary andpapillary thyroid carcinoma), renal carcinoma, kidney parenchymacarcinoma, cervix carcinoma, uterine corpus carcinoma, endometriumcarcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma,melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma,medulloblastoma and peripheral neuroectodermal tumors, gall bladdercarcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma,retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma,craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma,liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.

In a further embodiment, the cancer may be a lung cancer includingnon-small cell lung cancer and small cell lung cancer (including smallcell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma,and combined small cell carcinoma), colon cancer, breast cancer,prostate cancer, liver cancer, pancreas cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma,hepatocellular carcinoma, papillary renal carcinoma, head and necksquamous cell carcinoma, leukemia, lymphoma, myeloma, or a solid tumor.

In embodiments, the cancer comprises an acute lymphoblastic leukemia;acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers;AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas;atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer;brain stem glioma; brain tumor (including brain stem glioma, centralnervous system atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma); breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site; carcinoidtumor; carcinoma of unknown primary site; central nervous systematypical teratoid/rhabdoid tumor; central nervous system embryonaltumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer;medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma;Merkel cell skin carcinoma; mesothelioma; metastatic squamous neckcancer with occult primary; mouth cancer; multiple endocrine neoplasiasyndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm;mycosis fungoides; myelodysplastic syndromes; myeloproliferativeneoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma;Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lungcancer; oral cancer; oral cavity cancer; oropharyngeal cancer;osteosarcoma; other brain and spinal cord tumors; ovarian cancer;ovarian epithelial cancer; ovarian germ cell tumor; ovarian lowmalignant potential tumor; pancreatic cancer; papillomatosis; paranasalsinus cancer; parathyroid cancer; pelvic cancer; penile cancer;pharyngeal cancer; pineal parenchymal tumors of intermediatedifferentiation; pineoblastoma; pituitary tumor; plasma cellneoplasm/multiple myeloma; pleuropulmonary blastoma; primary centralnervous system (CNS) lymphoma; primary hepatocellular liver cancer;prostate cancer; rectal cancer; renal cancer; renal cell (kidney)cancer; renal cell cancer; respiratory tract cancer; retinoblastoma;rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small celllung cancer; small intestine cancer; soft tissue sarcoma; squamous cellcarcinoma; squamous neck cancer; stomach (gastric) cancer;supratentorial primitive neuroectodermal tumors; T-cell lymphoma;testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroidcancer; transitional cell cancer; transitional cell cancer of the renalpelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer;uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer;Waldenström macroglobulinemia; or Wilm's tumor.

The methods of the invention can be used to determine biomarker patternsor biomarker signature sets in a number of tumor types, diseased tissuetypes, or diseased cells including accessory, sinuses, middle and innerear, adrenal glands, appendix, hematopoietic system, bones and joints,spinal cord, breast, cerebellum, cervix uteri, connective and softtissue, corpus uteri, esophagus, eye, nose, eyeball, fallopian tube,extrahepatic bile ducts, other mouth, intrahepatic bile ducts, kidney,appendix-colon, larynx, lip, liver, lung and bronchus, lymph nodes,cerebral, spinal, nasal cartilage, excl. retina, eye, nos, oropharynx,other endocrine glands, other female genital, ovary, pancreas, penis andscrotum, pituitary gland, pleura, prostate gland, rectum renal pelvis,ureter, peritonem, salivary gland, skin, small intestine, stomach,testis, thymus, thyroid gland, tongue, unknown, urinary bladder, uterus,nos, vagina & labia, and vulva, nos.

In some embodiments, the molecular profiling methods are used toidentify a treatment for a cancer of unknown primary (CUP).Approximately 40,000 CUP cases are reported annually in the US. Most ofthese are metastatic and/or poorly differentiated tumors. Becausemolecular profiling can identify a candidate treatment depending onlyupon the diseased sample, the methods of the invention can be used inthe CUP setting. Moreover, molecular profiling can be used to createsignatures of known tumors, which can then be used to classify a CUP andidentify its origin. In an aspect, the invention provides a method ofidentifying the origin of a CUP, the method comprising performingmolecular profiling on a panel of diseased samples to determine a panelof molecular profiles that correlate with the origin of each diseasedsample, performing molecular profiling on a CUP sample, and correlatingthe molecular profile of the CUP sample with the molecular profiling ofthe panel of diseased samples, thereby identifying the origin of the CUPsample. The identification of the origin of the CUP sample can be madeby matching the molecular profile of the CUP sample with the molecularprofiles that correlate most closely from the panel of disease samples.The molecular profiling can use any of the techniques described herein,e.g., IHC, FISH, microarray and sequencing. The diseased samples and CUPsamples can be derived from a patient sample, e.g., a biopsy sample,including a fine needle biopsy. In one embodiment, DNA microarray andIHC profiling are performed on the panel of diseased samples, DNAmicroarray is performed on the CUP samples, and then IHC is performed onthe CUP sample for a subset of the most informative genes as indicatedby the DNA microarray analysis. This approach can identify the origin ofthe CUP sample while avoiding the expense of performing unnecessary IHCtesting. The IHC can be used to confirm the microarray findings.

The biomarker patterns or biomarker signature sets of the cancer ortumor can be used to determine a therapeutic agent or therapeuticprotocol that is capable of interacting with the biomarker pattern orsignature set. For example, with advanced breast cancer,immunohistochemistry analysis can be used to determine one or more geneexpressed proteins that are overexpressed. Accordingly, a biomarkerpattern or biomarker signature set can be identified for advanced stagebreast cancer and a therapeutic agent or therapeutic protocol can beidentified which is capable of interacting with the biomarker pattern orsignature set.

These examples of biomarker patterns or biomarker signature sets foradvanced stage breast cancer are just one example of the extensivenumber of biomarker patterns or biomarker signature sets for a number ofadvanced stage diseases or cancers that can be identified from thetables depicted in FIGS. 26-31. In addition, a number of non diseasespecific therapies or therapeutic protocols may be identified fortreating patients with these biomarker patterns or biomarker signaturesets by using method steps of the present invention described above suchas depicted in FIGS. 1-2 and FIGS. 5-14.

The biomarker patterns and/or biomarker signature sets disclosed in thetable depicted in FIGS. 26 and 28, and the tables depicted in FIGS. 27and 30 may be used for a number of purposes including, but not limitedto, specific cancer/disease detection, specific cancer/diseasetreatment, and identification of new drug therapies or protocols forspecific cancers/diseases. The biomarker patterns and/or biomarkersignature sets disclosed in the table depicted in FIGS. 26 and 28, andthe tables depicted in FIGS. 27 and 30 can also represent drug resistantexpression profiles for the specific tumor type or cancer type. Thebiomarker patterns and/or biomarker signature sets disclosed in thetable depicted in FIGS. 26 and 28, and the tables depicted in FIGS. 27and 30 represent advanced stage drug resistant profiles.

The biomarker patterns and/or biomarker signature sets can comprise atleast one biomarker. In yet other embodiments, the biomarker patterns orsignature sets can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10biomarkers. In some embodiments, the biomarker signature sets orbiomarker patterns can comprise at least 15, 20, 30, 40, 50, or 60biomarkers. In some embodiments, the biomarker signature sets orbiomarker patterns can comprise at least 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or50,000 biomarkers. Analysis of the one or more biomarkers can be by oneor more methods. For example, analysis of 2 biomarkers can be performedusing microarrays. Alternatively, one biomarker may be analyzed by IHCand another by microarray. Any such combinations of methods andbiomarkers are contemplated herein.

The one or more biomarkers can be selected from the group consisting of,but not limited to: Her2/Neu, ER, PR, c-kit, EGFR, MLH1, MSH2, CD20,p53, Cyclin D1, bcl2, COX-2, Androgen receptor, CD52, PDGFR, AR, CD25,VEGF, HSP90, PTEN, RRM1, SPARC, Survivin, TOP2A, BCL2, HIF1A, AR, ESR1,PDGFRA, KIT, PDGFRB, CDW52, ZAP70, PGR, SPARC, GART, GSTP1, NFKBIA,MSH2, TXNRD1, HDAC1, PDGFC, PTEN, CD33, TYMS, RXRB, ADA, TNF, ERCC3,RAF1, VEGF, TOP1, TOP2A, BRCA2, TK1, FOLR2, TOP2B, MLH1, IL2RA, DNMT1,HSPCA, ERBR2, ERBB2, SSTR1, VHL, VDR, PTGS2, POLA, CES2, EGFR, OGFR,ASNS, NFKB2, RARA, MS4A1, DCK, DNMT3A, EREG, Epiregulin, FOLR1, GNRH1,GNR1ER1, FSHB, FSHR, FSHPRH1, folate receptor, HGF, HIG1, IL13RA1, LTB,ODC1, PPARG, PPARGC1, Lymphotoxin Beta Receptor, Myc, Topoisomerase II,TOPO2B, TXN, VEGFC, ACE2, ADH1C, ADH4, AGT, AREG, CA2, CDK2, caveolin,NFKB1, ASNS, BDCA1, CD52, DHFR, DNMT3B, EPHA2, FLT1, HSP90AA1, KDR, LCK,MGMT, RRM1, RRM2, RRM2B, RXRG, SRC, SSTR2, SSTR3, SSTR4, SSTR5, VEGFA,or YES1.

For example, a biological sample from an individual can be analyzed todetermine a biomarker pattern or biomarker signature set that comprisesa biomarker such as HSP90, Survivin, RRM1, SSTRS3, DNMT3B, VEGFA, SSTR4,RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR,KDR, EPHA2, RXRG, or LCK. In other embodiments, the biomarker SPARC,HSP90, TOP2A, PTEN, Survivin, or RRM1 forms part of the biomarkerpattern or biomarker signature set. In yet other embodiments, thebiomarker MGMT, SSTRS3, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B,HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG,CD52, or LCK is included in a biomarker pattern or biomarker signatureset. In still other embodiments, the biomarker hENT1, cMet, P21, PARP-1,TLE3 or IGF1R is included in a biomarker pattern or biomarker signatureset.

The expression level of HSP90, Survivin, RRM1, SSTRS3, DNMT3B, VEGFA,SSTR4, RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1,DHFR, KDR, EPHA2, RXRG, or LCK can be determined and used to identify atherapeutic for an individual. The expression level of the biomarker canbe used to form a biomarker pattern or biomarker signature set.Determining the expression level can be by analyzing the levels of mRNAor protein, such as by microarray analysis or IHC. In some embodiments,the expression level of a biomarker is performed by IHC, such as forSPARC, TOP2A, or PTEN, and used to identify a therapeutic for anindividual. The results of the IHC can be used to form a biomarkerpattern or biomarker signature set. In yet other embodiments, abiological sample from an individual or subject is analyzed for theexpression level of CD52, such as by determining the mRNA expressionlevel by methods including, but not limited to, microarray analysis. Theexpression level of CD52 can be used to identify a therapeutic for theindividual. The expression level of CD52 can be used to form a biomarkerpattern or biomarker signature set. In still other embodiments, thebiomarkers hENT1, cMet, P21, PARP-1, TLE3 and/or IGF1R are assessed toidentify a therapeutic for the individual.

As described herein, the molecular profiling of one or more targets canbe used to determine or identify a therapeutic for an individual. Forexample, the expression level of one or more biomarkers can be used todetermine or identify a therapeutic for an individual. The one or morebiomarkers, such as those disclosed herein, can be used to form abiomarker pattern or biomarker signature set, which is used to identifya therapeutic for an individual. In some embodiments, the therapeuticidentified is one that the individual has not previously been treatedwith. For example, a reference biomarker pattern has been establishedfor a particular therapeutic, such that individuals with the referencebiomarker pattern will be responsive to that therapeutic. An individualwith a biomarker pattern that differs from the reference, for examplethe expression of a gene in the biomarker pattern is changed ordifferent from that of the reference, would not be administered thattherapeutic. In another example, an individual exhibiting a biomarkerpattern that is the same or substantially the same as the reference isadvised to be treated with that therapeutic. In some embodiments, theindividual has not previously been treated with that therapeutic andthus a new therapeutic has been identified for the individual.

Molecular profiling according to the invention can take on abiomarker-centric or a therapeutic-centric point of view. Although theapproaches are not mutually exclusive, the biomarker-centric approachfocuses on sets of biomarkers that are expected to be informative for atumor of a given tumor lineage, whereas the therapeutic-centric pointapproach identifies candidate therapeutics using biomarker panels thatare lineage independent. In a biomarker-centric view, panels of specificbiomarkers are run on different tumor types. See FIG. 46A. This approachprovides a method of identifying a candidate therapeutic by collecting asample from a subject with a cancer of known origin, and performingmolecular profiling on the cancer for specific biomarkers depending onthe origin of the cancer. The molecular profiling can be performed usingany of the various techniques disclosed herein. As an example, FIG. 46Ashows biomarker panels for breast cancer, ovarian cancer, colorectalcancer, lung cancer, and a “complete” profile to run on any cancer. Inthe figure, markers shown in italics are assessed using mutationalanalysis (e.g., sequencing approaches), marker shown underlined areanalyzed by FISH, and the remainder are analyzed using IHC. DNAmicroarray profiling can be performed on any sample. The candidatetherapeutic is selected based on the molecular profiling resultsaccording to the subject methods. An advantage to the bio-marker centricapproach is only performing assays that are most likely to yieldinformative results. Another advantage is that this approach can focuson identifying therapeutics conventionally used to treat cancers of thespecific lineage. In a therapeutic-centric approach, the biomarkersassessed are not dependent on the origin of the tumor. See FIG. 46B.This approach provides a method of identifying a candidate therapeuticby collecting a sample from a subject with a cancer, and performingmolecular profiling on the cancer for a panel of biomarkers withoutregards to the origin of the cancer. The molecular profiling can beperformed using any of the various techniques disclosed herein. As anexample, in FIG. 46B, markers shown in italics are assessed usingmutational analysis (e.g., sequencing approaches), marker shownunderlined are analyzed by FISH, and the remainder are analyzed usingIHC. DNA microarray profiling can be performed on any sample. Thecandidate therapeutic is selected based on the molecular profilingresults according to the subject methods. An advantage to thetherapeutic-marker centric approach is that the most promisingtherapeutics are identified only taking into account the molecularcharacteristics of the tumor itself. Another advantage is that themethod can be preferred for a cancer of unidentified primary origin(CUP). In some embodiments, a hybrid of biomarker-centric andtherapeutic-centric points of view is used to identify a candidatetherapeutic. This method comprises identifying a candidate therapeuticby collecting a sample from a subject with a cancer of known origin, andperforming molecular profiling on the cancer for a comprehensive panelof biomarkers, wherein a portion of the markers assessed depend on theorigin of the cancer. For example, consider a breast cancer. Acomprehensive biomarker panel is run on the breast cancer, e.g., thecomplete panel as shown in FIG. 46B, but additional sequencing analysisis performed on one or more additional markers, e.g., BRCA1 or any othermarker with mutations informative for theranosis or prognosis of thebreast cancer. Theranosis can be used to refer to the likely efficacy ofa therapeutic treatment. Prognosis refers to the likely outcome of anillness. One of skill will apprecitate that the hybrid approach can beused to identify a candidate therapeutic for any cancer havingadditional biomarkers that provide theranostic or prognosticinformation, including the cancers disclosed herein.

Methods for providing a theranosis of disease include selectingcandidate therapeutics for various cancers by assessing a sample from asubject in need thereof (i.e., suffering from a particular cancer). Thesample is assessed by performing an immunohistochemistry (IHC) todetermine of the presence or level of: AR, BCRP, c-KIT, ER, ERCC1, HER2,IGF1R, MET (also referred to herein as cMet), MGMT, MRP1, PDGFR, PGP,PR, PTEN, RRM1, SPARC, TOPO1, TOP2A, TS, COX-2, CK5/6, CK14, CK17, Ki67,p53, CAV-1, CYCLIN D1, EGFR, E-cadherin, p95, TLE3 or a combinationthereof; performing a microarray analysis on the sample to determine amicroarray expression profile on one or more (such as at least five, 10,15, 20, 25, 30, 40, 50, 60, 70 or all) of: ABCC1, ABCG2, ADA, AR, ASNS,BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1,DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1,FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3,IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2,NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR,POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG,SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1,TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; comparingthe results obtained from the IHC and microarray analysis against arules database, wherein the rules database comprises a mapping ofcandidate treatments whose biological activity is known against a cancercell that expresses one or more proteins included in the IHC expressionprofile and/or expresses one or more genes included in the microarrayexpression profile; and determining a candidate treatment if thecomparison indicates that the candidate treatment has biologicalactivity against the cancer.

Assessment can further comprise determining a fluorescent in-situhybridization (FISH) profile of EGFR, HER2, cMYC, TOP2A, MET, or acombination thereof, comparing the FISH profile against a rules databasecomprising a mapping of candidate treatments predetermined as effectiveagainst a cancer cell having a mutation profile for EGFR, HER2, cMYC,TOP2A, MET, or a combination thereof, and determining a candidatetreatment if the comparison of the FISH profile against the rulesdatabase indicates that the candidate treatment has biological activityagainst the cancer.

As explained further herein, the FISH analysis can be performed based onthe origin of the sample. This can avoid unnecessary laboratoryprocedures and concomitant expenses by targeting analysis of genes thatare known to play a role in a particular disorder, e.g., a particulartype of cancer. In an embodiment, EGFR, HER2, cMYC, and TOP2A areassessed for breast cancer. In another embodiment, EGFR and MET areassessed for lung cancer. Alternately, FISH analysis of all of EGFR,HER2, cMYC, TOP2A, MET can be performed on a sample. The complete panelmay be assessed, e.g., when a sample is of unknown or mixed origin, toprovide a comprehensive view of an unusual sample, or when economies ofscale dictate that it is more efficient to perform FISH on the entirepanel than to make individual assessments.

In an additional embodiment, the sample is assessed by performingnucleic acid sequencing on the sample to determine a presence of amutation of KRAS, BRAF, NRAS, PIK3CA (also referred to as PI3K), c-Kit,EGFR, or a combination thereof, comparing the results obtained from thesequencing against a rules database comprising a mapping of candidatetreatments predetermined as effective against a cancer cell having amutation profile for KRAS, BRAF, NRAS, PIK3CA, c-Kit, EGFR, or acombination thereof; and determining a candidate treatment if thecomparison of the sequencing to the mutation profile indicates that thecandidate treatment has biological activity against the cancer.

As explained further herein, the nucleic acid sequencing can beperformed based on the origin of the sample. This can avoid unnecessarylaboratory procedures and concomitant expenses by targeting analysis ofgenes that are known to play a role in a particular disorder, e.g., aparticular type of cancer. In an embodiment, the sequences of PIK3CA andc-KIT are assessed for breast cancer. In another embodiment, thesequences of KRAS and BRAF are assessed for GI cancers such ascolorectal cancer. In still another embodiment, the sequences of KRAS,BRAF and EGFR are assessed for lung cancer. Alternately, sequencing ofall of KRAS, BRAF, NRAS, PIK3CA, c-Kit, EGFR can be performed on asample. The complete panel may be sequenced, e.g., when a sample is ofunknown or mixed origin, to provide a comprehensive view of an unusualsample, or when economies of scale dictate that it is more efficient tosequence the entire panel than to make individual assessments.

The genes and gene products used for molecular profiling, e.g., bymicroarray, IHC, FISH, sequencing, and/or PCR, can be selected fromthose listed in Table 2. In an embodiment, INC is performed for one ormore, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, of: AR, BCRP,CAV-1, CD20, CD52, CK 5/6, CK14, CK17, c-kit, CMET, COX-2, Cyclin D1,E-Cad, EGFR, ER, ERCC1, HER-2, IGF1R, Ki67, MGMT, MRP1, P53, p95, PDGFR,PGP, PR, PTEN, RRM1, SPARC, TLE3, TOPO1, TOPO2A, TS, TUBB3; microarrayanalysis is performed on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 40, 50 or more, of: ABCC1, ABCG2, ADA, AR, ASNS, BCL2,BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, cKit, c-MYC, DCK, DHFR,DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERCC1, ERCC3, ESR1, FLT1,FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HER2/ERBB2, HIF1A, HSP90,IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, LCK, LYN, MET, MGMT, MLH1, MS4A1,MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRa, PDGFRA, PDGFRB,PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG,SIK2, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, SPARC, TK1, TNF, TOP2B,TOP2A, TOPO1, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70;fluorescent in-situ hybridization (FISH) is performed on 1, 2, 3, 4, 5,6 or 7 of ALK, cMET, c-MYC, EGFR, HER-2, PIK3CA, and TOPO2A; and DNAsequencing or PCR are performed on 1, 2, 3, 4, 5 or 6 of BRAF, c-kit,EGFR, KRAS, NRAS, and PIK3CA. In an embodiment, all of these genesand/or the gene products thereof are assessed.

Assessing one or more biomarkers disclosed herein can be used forcharacterizing any of the cancers disclosed herein. Characterizingincludes the diagnosis of a disease or condition, the prognosis of adisease or condition, the determination of a disease stage or acondition stage, a drug efficacy, a physiological condition, organdistress or organ rejection, disease or condition progression,therapy-related association to a disease or condition, or a specificphysiological or biological state.

A cancer in a subject can be characterized by obtaining a biologicalsample from a subject and analyzing one or more biomarkers from thesample. For example, characterizing a cancer for a subject or individualmay include detecting a disease or condition (including pre-symptomaticearly stage detecting), determining the prognosis, diagnosis, ortheranosis of a disease or condition, or determining the stage orprogression of a disease or condition. Characterizing a cancer can alsoinclude identifying appropriate treatments or treatment efficacy forspecific diseases, conditions, disease stages and condition stages,predictions and likelihood analysis of disease progression, particularlydisease recurrence, metastatic spread or disease relapse. Characterizingcan also be identifying a distinct type or subtype of a cancer. Theproducts and processes described herein allow assessment of a subject onan individual basis, which can provide benefits of more efficient andeconomical decisions in treatment.

In an aspect, characterizing a cancer includes predicting whether asubject is likely to respond to a treatment for the cancer. As usedherein, a “responder” responds to or is predicted to respond to atreatment and a “non-responder” does not respond or is predicted to notrespond to the treatment. Biomarkers can be analyzed in the subject andcompared to biomarker profiles of previous subjects that were known torespond or not to a treatment. If the biomarker profile in a subjectmore closely aligns with that of previous subjects that were known torespond to the treatment, the subject can be characterized, orpredicted, as a responder to the treatment. Similarly, if the biomarkerprofile in the subject more closely aligns with that of previoussubjects that did not respond to the treatment, the subject can becharacterized, or predicted as a non-responder to the treatment.

The sample used for characterizing a cancer can be any disclosed herein,including without limitation a tissue sample, tumor sample, or a bodilyfluid. Bodily fluids that can be used included without limitationperipheral blood, sera, plasma, ascites, urine, cerebrospinal fluid(CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor,amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid,semen (including prostatic fluid), Cowper's fluid or pre-ejaculatoryfluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid,pleural and peritoneal fluid, pericardial fluid, malignant effusion,lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,vomit, vaginal secretions, mucosal secretion, stool water, pancreaticjuice, lavage fluids from sinus cavities, bronchopulmonary aspirates orother lavage fluids. In an embodiment, the sample comprises vesicles.The biomarkers can be associated with the vesicles. In some embodiments,vesicles are isolated from the sample and the biomarkers associated withthe vesicles are assessed.

Pancreatic Cancer

For all stages of pancreatic cancer combined, the 1- and 5-year relativesurvival rates are 24% and 5% respectively. Even for those diagnosedwith local disease, the 5-year survival rate is only 20%. (AmericanCancer Society. (2009). Cancer Facts & Figures 2009. Atlanta: AmericanCancer Society. p. 19.) Target Now is a test that helps determine thestatus of a subject's molecular profile relevant to pancreatic cancerand delivers a single evidence—based report with individualizedtherapeutic guidance. Because so many pancreatic cancer patients getjust one chance for chemotherapy, molecular profiling can provide theinformation needed to make an appropriate first choice.

Molecular profiling according to the methods of the invention can beused to make informed treatment decisions for pancreatic cancerpatients, including without limitation those who are eligible forsystemic treatment, or have progressed on prior therapy.

Therapeutic agents that can be associated with clinical benefit or lackof clinical benefit based on biomarker status include Anti-NeoplasticAgent (gemcitabine), Platinum Analogues (cisplatin, oxaliplatin),Protein Kinase Inhibitor (erlotinib), Pyrimidine Analogues(5-fluorouracil, capecitabine), Taxane (nab-paclitaxel).

For a sample from a subject suffering from pancreatic cancer, IHCprofiling can be conducted to determine the presence or level of one ormore: AR, BCRP, c-KIT, ER, ERCC1, HER2, MGMT, MRP1, PDGFR, PGP, PR,PTEN, RRM1, SPARC, TOPO1, TOP2A, and TS. In some embodiments, IHC isconducted on all of these biomarkers. The IHC analysis can be combinedwith microarray analysis, as described further herein. The analysis canfurther comprise assessing EGFR, HER2 or both by FISH and/or nucleicacid sequencing of KRAS, BRAF, or both. In another embodiment, molecularprofiling performed on a sample from a subject with pancreatic cancerincludes the tests listed in Table 7. Based on results for one or moreof the foregoing (i.e., IHC, FISH, sequencing, microarray), a treatmentor therapy is selected. Based on the analysis, a likelihood of clinicalbenefit or lack of clinical benefit of a particular candidate treatmentis determined. Illustrative treatments include without limitation ananti-neoplastic, platinum analog, protein kinase inhibitor, pyrimidineanalog, or a taxane, or any combination thereof, such as gemcitabine,cisplatin, oxaliplatin, erlotinib, 5-fluorouracil, capecitabine, ornab-paclitaxel. In some embodiments, the subject assessed withpancreatic cancer is eligible for systemic treatment or has beensubjected to prior therapy.

TABLE 7 Molecular Profiling for Pancreatic Cancer: Biomarkers AssessedIHC AR PGP BCRP PR c-KIT PTEN ER RRM1 ERCC1 SPARC HER2 Mono MGMT SPARCPoly MRP1 TOPO1 PDGFR TOP2A TS FISH   EGFR (if appropriate)   HER2 (ifappropriate) Mutation Analysis   BRAF (if appropriate)   KRAS (ifappropriate) DNA Microarray   Whole genome expression array

Lung Cancer

The 1-year relative survival for lung cancer is 41%. The 5-year survivalrate for all stages combined is only 15%. The 5-year survival rate is50% for cases detected when the disease is localized, but only 16% oflung cancers are diagnosed at this early stage. Lung cancer patientsoften present with advanced disease, which is a major treatmentchallenge. Their performance status precludes using many toxicchemotherapies making initial treatment selection critical. (AmericanCancer Society. (2009). Cancer Facts & Figures 2009. Atlanta: AmericanCancer Society. p. 15.)

Molecular profiling according to the methods of the invention resultscan be used to make informed treatment decisions for lung cancerpatients, including without limitation those who have non-small celllung cancer (NSCLC) with stage 1V metastatic disease who have progressedthrough platinum combination regimens and now require select second-linetherapies (and beyond), or want to guide first-line therapy for NSCLCwet stage Mb and Stage 1V disease, or have small cell lung cancer (SCLC)and have failed first line therapy, or have mesothelioma and have failedfirst line therapy.

Therapeutic agents that can be associated with clinical benefit or lackof clinical benefit based on biomarker status include Taxanes(paclitaxel, docetaxel, nab-paclitaxel), Vinca Alkyloids (vinblastine,vinorelbine), Anti-Neoplastic Agents (gemcitabine, mitomycin),Podophyllotoxin Derivative (etoposide), Anti-Vascular Agent(bevacizumab), Platinum Analogues (carboplatin, cisplatin),Podophyllotoxin Derivative (etoposide).

For a sample from a subject suffering from lung cancer, IHC profilingcan be conducted to determine the presence or level of one or more of:AR, BCRP, c-KIT, ER, ERCC1, IGF1R, HER2, MET, MGMT, MRP1, PDGFR, PGP,PR, PTEN, RRM1, SPARC, TOPO1, TOP2A, and TS. In some embodiments, IHC isconducted on all of these biomarkers. The IHC analysis can be combinedwith microarray analysis. In some embodiments, the analysis furthercomprises nucleic acid sequencing of EGFR. The analysis can furthercomprise assessing one or more of EGFR, HER2 and MET by FISH and/ornucleic acid sequencing of one or more of KRAS, BRAF, and EGFR. In someembodiments, EGFR and MET are analyzed by FISH. In some embodiments,KRAS, BRAF, and EGFR are analyzed by nucleic acid sequencing. In someembodiments, molecular profiling of a lung cancer is performed todetermine the presence, level or mutation in one or more of EML4-ALK,C-MET, Beta III tubulins, EGFR mutation (e.g., by FISH), PTEN, KRAS,BRAF, ERCC1, MRP1, BCRP, PGP, TS, RRM1, TOP2A, TOPO1, and COX2. Inanother embodiment, molecular profiling performed on a sample from asubject with lung cancer includes the tests listed in Table 8. Based onresults for one or more of the foregoing (i.e., IHC, FISH, sequencing,microarray), a candidate treatment or therapy is selected. Based on theanalysis, a likelihood of clinical benefit or lack of clinical benefitof a particular candidate treatment is determined. Illustrativetreatments include without limitation a taxane, a vinca alkyloid,anti-neoplastic agent, podophyllotoxin derivative, anti-vascular agent,platinum analog, protein kinase inhibitor, folic acid analog,topoisomerase inhibitor, monoclonal antibody, or a or any combinationthereof, such as paclitaxel, docetaxel, nab-paclitaxel, vinblastine,vinorelbine, gemcitabine, mitomycin, etoposide, bevacizumab,carboplatin, cisplatin, erlotinib, gefitinib, anthracycline,doxorubicin, pemetrexed, topotecan, irinotecan, or cetuximab. Thesubject may have non-small cell lung cancer (NSCLC), small cell lungcancer (SCLC), or mesothelioma. In another embodiment, the subject hasNSCLC with stage 1V metastatic disease and has progressed throughplatinum combination regimens. In yet another embodiment, the subjecthas NSCLC wet Stage Mb and Stage 1V disease. In one embodiment, thesubject has failed first line therapy.

TABLE 8 Molecular Profiling for Lung Cancer: Biomarkers Assessed IHC ARPDGFR BCRP PGP c-KIT PR ER PTEN ERCC1 RRM1 HER2 SPARC Mono IGF1R SPARCPoly MET TOPO1 MGMT TOP2A MRP1 TS FISH EGFR (if appropriate) HER2 (ifappropriate) MET (if appropriate) Mutation Analysis Mutation AnalysisEGFR BRAF (if appropriate) KRAS (if appropriate) DNA Microarray Wholegenome expression array

Colorectal Cancer

Colorectal cancer is the second leading cause of cancer death in theUnited States. The 1- and 5-year relative survival for persons withcolorectal cancer is 83% and 64%, respectively. The 5-year survival ratedrops to 68% after cancer has spread to involve adjacent organs andlymph nodes. For persons with distant metastases, 5-year survival is11%. The NCCN guidelines state that patients who are KRAS and BRAFmutated are not likely to respond to EGFR-inhibiting therapies andshould receive alternative treatment. (American Cancer Society. (2009).Cancer Facts & Figures 2009. Atlanta: American Cancer Society. p.12-13.)

Molecular profiling according to the methods of the invention can beused to make informed treatment decisions for colorectal cancerpatients, including without limitation those who have been treated formetastatic disease and have progressed, or have disease that isrefractory to standard of care and for whom no clear treatment optionsexist.

Therapeutic agents that can be associated with clinical benefit or lackof clinical benefit based on biomarker status include Anti-VascularAgent (bevacizumab), Monoclonal Antibodies (cetuximab, panitumumab),Platinum Analogue (oxaliplatin), Pyrimidine Analogues (5-fluorouracil,capecitabine), Topoisomerase Inhibitor (irinotecan).

For a sample from a subject suffering from colon cancer or colorectalcancer, IHC profiling can be conducted to determine the presence orlevel of one or more of: COX-2, PTEN, TOP1, TOP2A and TS. In someembodiments, IHC is conducted on all of these biomarkers. The IHCanalysis can be combined with microarray analysis and/or nucleic acidsequencing of KRAS, BRAF, or both. The subject may colorectal coloncancer that is non-metastatic or treatment-naive metastatic.Alternately, the subject has colorectal cancer that is metastatic or thesubject has failed prior therapy. IHC can be performed on additionalbiomarkers, such as one or more of: AR, BCRP, c-KIT, ER, ERCC1, HER2,MGMT, MRP1, PDGFR, PGP, PR, RRM1, and SPARC. In some embodiments, IHC isconducted on all of these additional biomarkers. The analysis canfurther comprise assessing HER2 by FISH. In another embodiment,molecular profiling performed on a sample from a subject with colorectalcancer includes the tests listed in Table 9. Based on results for one ormore of the foregoing (i.e., IHC, FISH, sequencing, microarray), atreatment or therapy is selected. Based on the analysis, a likelihood ofclinical benefit or lack of clinical benefit of a particular candidatetreatment is determined. Illustrative treatments include withoutlimitation an anti-vascular agent, monoclonal antibody, platinum analog,pyrimidine analog, topoisomerase inhibitor, or any combination thereof,such as bevacizumab, cetuximab, panitumumab, oxaliplatin,5-fluorouracil, capecitabine, or irinotecan. The subject can be asubject that has been treated for metastatic colorectal cancer that hasprogressed, can be currently treated for metastatic colorectal cancerthat has progressed, and/or has disease that is refractory to a standardof care. In another embodiment, the subject has no clear treatmentoptions.

TABLE 9 Molecular Profiling for Colorectal Cancer: Biomarkers AssessedNon-metastatic or treatment-naïve metastatic Metastatic and failed priortherapy IHC IHC COX-2 AR ERCC1 PGP SPARC PTEN BCRP HER2 PR Mono TOPO1c-KIT MGMT PTEN SPARC TS COX-2 MRP1 RRM1 Poly ER PDGFR TOPO1 TOP2A TSFISH FISH NA HER2 (if appropriate) Mutation Analysis Mutation AnalysisBRAF BRAF KRAS KRAS DNA Microarray DNA Microarray Whole genome Wholegenome expression array expression array

Ovarian Cancer

Epithelial ovarian cancer is the most lethal gynecological cancer.Approximately 75% of women with ovarian cancer present with stage III/IVdisease. The estimated five year survival is 45% for all stages of thedisease, and 18.6% for stage 1V disease. See American Cancer Society.(2009). Cancer Facts & Figures 2009. Atlanta: American Cancer Society.p. 17-18. Risk factors that increase the risk for developing ovariancancers: older age at first birth pregnancy; nulliparity, the status ofa woman who has never borne a child; family history; genetic background,e.g., BRCA1/2 mutations; and long term hormone replacement therapy.

Ovarian surface epithelium carcinomas (EOC) make about 80% of ovariancancers. These include without limitation surface epithelial tumors,serous cancers, mucinous cancer, endometriod cancer, clear cell cancer,carcinosarcoma, Brenner tumors, and cancers of the fallopian tubes andfemale peritoneal cancers. Non-epithelial ovarian carcinomas (non-EOC)make up about 20% of ovarian cancers. These include sarcoma of theovary, malignant germ cell tumors, sex cord-stromal tumors,gonadoblastoma, and lymphomas and other rare tumors of the ovary.

Treatment strategies for ovarian cancers include surgery, chemotherapyand radiation therapy. Chemotherapy is chosen based on selectioncriteria comprising stage of disease and National Comprehensive CancerNetwork (NCCN) guidelines. First line treatment, or primary chemotherapyfor stages I-IV include: 1) cytotoxic therapy such as a platinum agent,e.g., Carbolatin/Cisplatin and a taxane, e.g., Paclitaxel or Docetaxel,and ii) targeted therapy such as bevacizumab, a humanized monoclonalantibody that recognizes and blocks vascular endothelial growth factor A(VEGF-A). Bevacizumab is sold under the trade name Avastin® by GenentechInc of South San Francisco, Calif., USA. Second line treatments afterprogression on first line primary treatments for stages II-IV depend onwhether the disease is platinum sensitive or platinum resistant.Preferred recurrence therapies for platinum sentitive disease includeplatinum/taxane combination, platinum and gemcitabine, platinum andliposome-entrapped doxorubincin (lip-doxorubicin), or single platinumagent therapy. Preferred recurrence therapies for platinum resistantdisease include docetaxel, etoposide, gemcitabine, lip-doxorubicin,weekly paclitaxel, pemetrexed or topotecan. Further information aboutNCCN guidelines is available at www.nccn.org.

Other potential active agents for second line use in recurrent ovariancancers include cytotoxic agents such as altretamine, capecitabine,cyclophosphamide, irinotecan, melphalan, oxaliplatin, paclitaxel, andvinorelbine; or hormonal agents such as anastrozole, letrozole,leuprolide acetate, megestrol acetate, and tamoxifen.

Because most patients with ovarian cancer have recurrent disease at somepoint, a proactive plan for deciding treatment options based on thepatient's tumor biology is an important aspect of care. Molecularprofiling can be used to make informed treatment decisions for ovariancancer patients, including without limitation those who have metastaticdisease, have progressed on platinum therapy, or have recurrent diseaseand have failed third line therapy. The invention provides methods ofmolecular profiling of ovarian cancers to guide the selection oftreatment, e.g., which chemotherapeutic agents are likely to beeffective, given the molecular characteristics of the tumor.

In an aspect, the invention provides a method of selecting a candidatetherapeutic for treating a patient with ovarian cancer, comprisingperforming molecular profiling on a sample from the subject. Molecularprofiling can take advantage of any appropriate methodology that can beused to characterize a biological sample. As disclosed herein, suchanalysis includes assessing the expression or mutational status of thetumor. Useful techniques include IHC, FISH, microarray, PCR andsequencing methods.

For a sample from a subject suffering from ovarian cancer, IHC profilingcan be conducted to determine the presence or level of one or more of:AR, BCRP, c-KIT, ER, ERCC1, HER2, MGMT, MRP1, PDGFR, PGP, PR, PTEN,RRM1, SPARC, TOPO1, TOP2A, and TS. In some embodiments, IHC is conductedon all of these biomarkers. In some embodiments, IHC profiling forovarian cancer is conducted to determine the presence or level of one ormore of: PGP, ER, TOPO1, TOP2A, ERCC1, TS, ER, PR, RRM1, BRCA1, BRCA2,PI3KCA, IGFRBP3, IGFRBP4, IGFRBP5, HER-2 and TLE3. In some embodiments,IHC is conducted on all of these biomarkers. The IHC analysis can becombined with microarray analysis. The analysis can further compriseassessing EGFR, HER2, or both by FISH and/or nucleic acid sequencing ofKRAS, BRAF, or both. Based on results for one or more of the foregoing(i.e., IHC, FISH, sequencing, microarray), a treatment or therapy isselected. Based on the analysis, a likelihood of clinical benefit orlack of clinical benefit of a particular candidate treatment isdetermined. Illustrative treatments include without limitation ananti-neoplastic, topoisomerase inhibitor, anthracycline, pyrimidineanalog, vinca alkaloid, podophyllotoxin derivative, taxane,anti-vascular agent, platinum analog, anti-estrogen therapy, aromataseinhibitor, folic acid analog, selective estrogen receptor modulator,gonadotropin releasing hormone analog or any combination thereof, suchas topotecan, irinotecan, gemcitabine, liposomal doxorubicin,capecitabine, vinblastine, vinorelbine, vincristine, etoposide,paclitaxel, docetaxel, bevacizumab, carboplatin, cisplatin, oxaliplatin,tamoxifen, fulvestrant, anastrozole, letrozole, megestrol, pemetrexed,tamoxifen, or leuprolide. In one embodiment, the subject has metastaticovarian cancer, has progressed on platinum therapy, or has recurrentdisease and has failed third line therapy.

In some embodiments, INC profiling for a sample from a subject sufferingfrom ovarian cancer is conducted to determine the presence or level ofone or more of: ER, HER2, Ki67, p53, PGP, PR, and TS. In someembodiments, INC is conducted on all of these biomarkers. The IHCanalysis can be combined with microarray analysis and/or assessing HER2by fluorescent in-situ hybridization (FISH). In another embodiment,molecular profiling performed on a sample from a subject with ovariancancer includes the tests listed in Table 10.

TABLE 10 Molecular Profiling for Ovarian Cancer: Biomarkers Assessed IHCAR MRP1 BCRA1 PDGFR BRCA2 PI3KCA BCRP PGP c-KIT PR ER PTEN ERCC1 RRM1HER2 SPARC Mono IGFRBP3 SPARC Poly IGFRBP4 TLE3 IGFRBP5 TOPO1 MGMT TOP2ATS FISH EGFR (if appropriate) HER2 (if appropriate) Mutation AnalysisBRAF (if appropriate) KRAS (if appropriate) DNA Microarray Whole genomeexpression array

The biomarkers assessed to identify a candidate therapeutic for ovariancancer can include one or more of ABCB1, BRCA 1, BRCA 2, CES2, cMET,DHFR, EGFR, ER, ERCC1, ESR1, GART, HER2, HIF-1a, IGFBP 3, IGFBP 4, IGFBP5, MGMT, MRP1, PGP, PIK3CA, PR, PTEN, RRM1, RRM2 and RRM2b, SPARC, SRC,TLE3, TOP2A, TOP2B, TOPO1, TUBB3, TYMS, VDR, VEGFR1, VEGFR2, and VHL. Inembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25or all of these markers is assessed. Methods of assessment are asdisclosed herein, e.g., microarray, IHC, ISH, FISH, and various forms ofsequencing and/or PCR based methods.

Therapeutic agents that can be associated with clinical benefit or lackof clinical benefit based on biomarker status of ovarian cancer includewithout limitation Topoisomerase Inhibitors (topotecan, irinotecan),Anti-Neoplastic Agent (gemcitabine), Anthracycline (liposomaldoxorubicin), Prymidine Analog (capecitabine), Vinca Alkaloids(vinblastine, vinorelbine, vincristine), Podophyllotoxin Derivative(etoposide), Taxanes (paclitaxel, docetaxel), Anti-Vascular Agent(bevacizumab), Platinum Analogues (carboplatin, cisplatin, oxaliplatin),Anti-Estrogen Therapy (tamoxifen, fulvestrant), Aromatase Inhibitors(anastrozole, letrozole, megestrol), Folic Acid Analogue (pemetrexed),Selective Estrogen Receptor Modulator (tamoxifen), GonadotropinReleasing Hormone Analogue (leuprolide). The therapeutic agent that canbe associated with clinical benefit or lack of clinical benefit based onbiomarker status of ovarian cancer further include without limitationaminoglutethimide, exemestane, anastrozole, letrozole, capecitabine,cisplatin, carboplatin, doxorubicin, liposomal-doxorubicin, gemcitabine,nab-paclitaxel, paclitaxel, docetaxel, pemetrexed, tamoxifen, topotecan,irinotecan, and/or trastuzumab.

The methods of the invention are used to select candidate therapeuticsincluding chemotherapeutic agents listed in Table 11. The candidatetherapeutics can be selected based on molecular profiling of theassociated genes listed in the table. As described herein, the profilingcan include expression analysis and/or mutational analysis of the genesat the nucleic acid and protein levels. For example, Table 11 providesillustrative methods used to assess the genes and/or gene productslisted therein. As described herein, multiple methods can be used toassess a biomarker, e.g., IHC and microarray. In such cases, rules canbe used to prioritize findings. In some embodiments, similar results forIHC and microarray, e.g., overexpression or underexpression compared tonormals, on a single gene/gene product has priority over a result withonly IHC or microarray. Similarly, IHC expression results can beprioritized over microarray expression results. The markers can beassociated with benefit or no-benefit of the agent.

TABLE 11 Candiate Agents and Associated Biomarkers for Ovarian CancerAgent Gene Method topotecan, irinotecan CES2 Microarray TOP1 MicroarrayTOPO1 IHC cisplatin, carboplatin BRCA1 Microarray BRCA2 Microarray ERCC1IHC Microarray oxaliplatin ERCC1 IHC Microarray gemcitabine RRM1 IHCMicroarray RRM2 Microarray RRM2B Microarray paclitaxel, docetaxel ABCB1Microarray PGP IHC TLE3 IHC doxorubicin, liposomal-doxorubicin TOP2A IHCMicroarray TOP2B Microarray etoposide TOP2A Microarray pemetrexed DHFRMicroarray GART Microarray TS IHC TYMS Microarray tamoxifen ER IHC ESR1Microarray PR IHC Microarray anastrozole, letrozole, aminoglutethimide,ER IHC exemestane ESR1 Microarray IGFBP3 Microarray IGFBP4 MicroarrayIGFBP5 Microarray PR IHC Microarray bevacizumab HIF-1α Microarray VEGFR2Microarray (KDR) VEGFR1 Microarray (FLT1) VHL Microarray nab-paclitaxelSPARC IHC Microarray erlotinib, gefitinib, cetuximab, panitumumab PTENIHC EGFR FISH trastuzumab HER2 FISH IHC mitomycin BRCA1 Microarray BRCA2Microarray temozolomide MGMT IHC Microarray dasatinib SRC Microarrayfulvestrant ER IHC ESR1 Microarray PR IHC Microarray gonadorelinAndrogen Microarray Receptor PR Microarray goserelin Androgen IHCReceptor Microarray PR Microarray sorafenib, sunitinib VEGFR1 MicroarrayVEGFR2 Microarray VHL Microarray toremifene ER IHC ESR1 Microarray PRIHC Microarray calcitriol, cholecalciferol VDR Microarray

Table 12 presents a number of genes whose expression or mutationalstatus can be used to select a candidate therapeutic agent.Overexpression or underexpression of the genes or their gene productscan be informative for selecting candidate agents and/or for avoidingagents that are likely to be ineffective (e.g., resistant agents). In anembodiment, the molecular profiling of an ovarian cancer sample includesanalysis of one or more gene, or a gene product thereof, listed in Table12. The expression of the gene or gene product is used to select acandidate agent or avoid an agent. The genes or gene products can beassessed using any appropriate technique described herein. In anembodiment, gene expression is analyzed by microarray, e.g., DNAmicroarray to assess mRNA expression. In an embodiment, gene expressionis analyzed at the protein level by IHC. The gene expression can also beanalyzed by other techniques, e.g., PCR or sequencing techniques.

TABLE 12 Expression Analysis for Ovarian Cancer Treatments Gene NameExpression Status Possible Agent(s) Possible Resistance BRCA1 UnderExpressed mitomycin, cisplatin, carboplatin BRCA2 Under Expressedmitomycin, cisplatin, carboplatin DHFR Under Expressed methotrexate,pemetrexed DHFR Over Expressed methotrexate ER Over Expressedanastrozole, exemestane, fulvestrant, letrozole, megestrol, tamoxifen,medroxyprogesterone, toremifene, aminoglutethimide ERCC1 Over ExpressedPlatinum-based therapy, carboplatin, cisplatin GART Under Expressedpemetrexed HER2 Over Expressed trastuzumab HIF-1α Over Expressedbevacizumab IGFBP3 Under Expressed letrozole IGFBP3 Over Expressedletrozole IGFBP4 Under Expressed letrozole IGFBP4 Over Expressedletrozole IGFBP5 Under Expressed letrozole IGFBP5 Over Expressedletrozole MGMT Under Expressed temozolomide MGMT Over Expressedtemozolomide P-gp (ABCB1) Over Expressed paclitaxel, docetaxel PR OverExpressed exemestane, fulvestrant, gonadorelin, goserelin,medroxyprogesterone, megestrol, tamoxifen, toremifene RRM1 UnderExpressed gemcitabine RRM2 Under Expressed gemcitabine RRM2B UnderExpressed gemcitabine SPARC Over Expressed nab-paclitaxel SRC OverExpressed dasatinib TLE-3 Over Expressed taxanes, paclitaxel, docetaxelTOPO I Over Expressed irinotecan, topotecan TOPO IIα Over Expresseddoxorubicin, liposomal- doxorubicin, etoposide TOPO IIβ Over Expresseddoxorubicin, liposomal- doxorubicin TS (TYMS) Under Expressed pemetrexedTS (TYMS) Over Expressed pemetrexed VDR Over Expressed calcitriol,cholecalciferol VEGFR1 (FLT1) Over Expressed sorafenib, sunitinib,bevacizumab VEGFR2 (KDR) Over Expressed sorafenib, sunitinib,bevacizumab VHL Under Expressed sorafenib, sunitinib, bevacizumab

In an aspect, the invention provides a method of identifying a candidatetreatment and/or a prognosis for a subject in need thereof by usingmolecular profiling of sets of known biomarkers. For example, the methodcan identify a chemotherapeutic agent for an individual with an ovariancancer. The method comprises: obtaining a sample from the subject;performing an immunohistochemistry (INC) analysis on the sample todetermine an IHC expression profile on one or more, e.g. 2, 3, 4, 5, 6,7, 8, 9, 10 or more, of: AR, ER, ERCC1, HER2, MGMT, PGP, PR, PTEN, RRM1,SPARC, TLE3, TOP2A, TOPO1, TS; performing a microarray analysis on thesample to determine a microarray expression profile on one or more, e.g.2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, of: BRCA1, BRCA2, DHFR, ER,ERCC1, GART, HIF-1α, IGFBP3, IGFBP4, IGFBP5, MGMT, P-gp (ABCB1), PR,RRM1, RRM2, RRM2B, SPARC, SRC, TOPO I, TOPO IIα, TOPO IIβ, TS (TYMS),VDR, VEGFR1 (FLT1), VEGFR2 (KDR), and WIT; and performing a fluorescentin-situ hybridization (FISH) analysis on the sample to determine a FISHmutation profile on one or more of EGFR and HER2. The method can furthercomprise comparing the IHC expression profile, microarray expressionprofile, FISH mutation profile and against a rules database, wherein therules database comprises a mapping of treatments whose biologicalactivity is known against diseased cells that: i) overexpress orunderexpress one or more proteins included in the IHC expressionprofile; ii) overexpress or underexpress one or more genes included inthe microarray expression profile; and/or iii) have zero or moremutations in one or more genes included in the FISH mutation profile;and identifying the treatment if the comparison against the rulesdatabase indicates that the treatment should have biological activityagainst the disease; and the comparison against the rules database doesnot contraindicate the treatment for treating the disease. The rules canbe those in Tables 5 or 12. The molecular profiling steps can beperformed in any order. In some embodiments, not all of the molecularprofiling steps are performed. As a non-limiting example, microarrayanalysis is not performed if the sample quality does not meet athreshold value, as described herein. In some embodiments, the IHCexpression profiling is performed on at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% of the gene products above. In some embodiments,the microarray expression profiling is performed on at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95% of the genes listed above. In someembodiments, the IHC expression profiling is performed on all of thegene products above. In some embodiments, the microarray profiling isperformed on all of the genes listed above. In some embodiments, theFISH profiling is performed on all of the gene products above.

The molecular profiling methods of the invention can be used to providea prognostic read out in addition to selecting candidate therapeuticagents. In such cases, molecular profiling of one or more biomarkers canprovide guidance for both therapeutic selection and prognosis. The oneor more biomarkers would be known to have an association with boththerapeutic efficacy and prognosis of a disease. Similarly, themolecular profiling can be performed on some markers that provideguidance for therapeutic selection, and other markers that guidance forprognostic outcome. In an embodiment, molecular profiling is performedon a sample from a subject to select one or more candidate therapeuticagent and to provide a prognosis. The markers used to select the one ormore select candidate therapeutic agent can include those listed inTables 11-12. The prognostic markers can include cMet, IGF1R, Class IIIbeta tubulin (TUBB3) and PIK3CA. cMet is commonly overexpressed inovarian cancers, where higher levels of cMet indicate later stagedisease and worse prognosis. Overexpression of cMet can also be used toindicate a subject for enrollment in a clinical trial of Met-targetingagents. Examples of Met-targeted agents include ARQ197, PF-02341066 andSCH900105. IGF1R and IGFBPs are a high affinity receptor forInsulin-like Growth Factor and binding proteins that regulate theeffects of IGF, respectively. The IGF axis is important for cellproliferation, differentiation and apoptosis in many cell types. Overexpression of IGF1R and IGFBPs associates with ovarian cancer risk ordisease progression. IGFBP and IGF1R expression can have predictiveutility for figitumumab. In addition, IGFBPs can help identifyestrogen-responsive patients. Tubulins are the building blocks formicrotubules which are filaments that form the cytoskeleton of the cell.Class 3 Beta isotype tubulin is associated with aggressive anddrug-resistant disease. High expression levels of PIII-Tubulinassociates with resistance to tubulin-binding agents, including taxaneslike paclitaxel. The PIK3CA gene encodes the catalytic subunit ofPI3-kinase. PIK3CA amplification is reported in 16-25% of ovariancancers. Increases in PIK3CA gene copy number leads to dysregulation ofother components of the pathway, including phosphorylation of AKT.Dysregulation of the AKT/PIK3CA pathway leads to cell proliferation,apoptosis and motility. PIK3CA amplification associates with resistanceto platinum and taxane combination therapy in ovarian cancer patients.Breast Cancer Susceptibility Genes 1/2 (BRCA1 and BRCA2) are tumorsuppressor proteins involved in repairing DNA. Mutation of BRCA1/2 leadsto impaired DNA repair which pre-disposes a person to breast and ovariancancers. Loss of expression or presence of mutations in BRCA1/2associates with response to platinum-based therapies. BRCA1/2 underexpression can associate with response to cisplatin or carboplatin, andmitomycin.

In an embodiment, cMet is assessed by IHC and/or FISH. In anotherembodiment, IGF1R and/or Class III beta tubulins are assessed by IHC. Instill another embodiment, PIK3CA is assessed by FISH. The expression ofcMet, IGF1R, Class III beta tubulin and/or PIK3CA can also be assessedusing a microarray. The microarray can be a DNA microarray which probesmRNA expression. In embodiments, one or more of cMet, IGF1R, Class IIIbeta tubulins and PIK3CA is assessed to provide a prognosis.

The present invention provides a method of selecting candidatetreatments for ovarian cancer sample by performing molecular profilinganalysis on a sample from a subject. In an embodiment, the cancer isreflexed depending on whether the cancer is an ovarian surfaceepithelium carcinoma (EOC) or non-epithelial ovarian cancer (non-EOC).In such cases, an EOC sample may not receive reflex testing when PTEN isnot negative, as assessed by IHC, and tests that will not be performedinclude: i) EGFR by FISH; ii) KRAS Mutational Analysis by TargetedSequencing; and iii) BRAF Mutational Analysis by Targeted Sequencing.Non-EOC will get reflexed when PTEN is not negative by IHC, includingassessment of EGFR by FISH. The profiling can include analysis of one ormore of the genes listed in Table 11 using the indicated profilingtechniques. The profiling can also use DNA microarray analysis of thegenes listed in Table 12. Profiling of cMet, IGF1R, Class III betatubulins and/or PIK3CA can also be performed. In an embodiment, cMet isassessed by IHC and/or FISH. In another embodiment, IGF1R and/or ClassIII beta tubulins are assessed by IHC. In still another embodiment,PIK3CA is assessed by FISH. The expression of cMet, IGF1R, Class IIIbeta tubulins and/or PIK3CA can also be assessed using a microarray. Theresults of the profiling analysis, e.g., expression and mutationanalysis, are used to select a candidate therapeutic agent and/orprovide a prognosis as described herein.

Breast Cancer

Breast cancer is the second most common type of cancer after lungcancer, and the fifth most common cause of cancer deaths. Althoughbreast cancer is 100-fold more prevalent in women, both sexes can beafflicted with the disease. Breast cancer usually starts in the breast,e.g., in the inner lining of the milk ducts or lobules. Various types ofbreast cancer are characterized by stage, aggressiveness and geneticevents. Treatments include surgery (e.g., mastectomy), drugs (hormonetherapy and chemotherapy, and radiation. 10 year survival ranges from 10to 98%. Non-invasive (or “in situ”) breast cancers are confined to ductsor lobules but can become invasive. Ductal carcinoma in situ (DCIS) isthe most common type of non-invasive breast cancer. Invasive (orinfiltrating) cancers have started to break through normal breast tissuebarriers and invade surrounding areas. Invasive cancers can be veryserious.

Some breast cancers require the hormones estrogen and progesterone toproliferate and express receptors for those hormones, e.g., the estrogenreceptor (ER) and progesterone receptor (PR). Such cancers can betreated with therapeutic agents that inhibit this process, e.g.,tamoxifen, an antagonist of the estrogen receptor in breast tissue, andaromatase inhibitors, which block the synthesis of estrogen. Interferingwith estrogen synthesis can damage the ovaries and lead to infertility.Breast cancers without hormone receptors, those that spread to the lymphnodes, or have other risk factors, may be treated more aggressively.“CA” therapy comprises a cocktail of cyclophosphamide and doxorubicin(Adriamycin®), which damage DNA. “CAT” therapy further includes a taxanedrug, such as docetaxel, which attacks microtubules. ‘CMF” therapycomprises cyclophosphamide, methotrexate, and fluorouracil. All of thesechemotherapeutic agents can cause serious side effects by affectingnormal cells. The HER2 gene (also known as HER2/neu and ErbB2 gene) isamplified in 20-30% of early-stage breast cancers. Trastuzumab(Herceptin™) is a monoclonal antibody that interferes with the HER2/neureceptor, thereby inhibiting cancer cell growth. Breast cancers thatdon't overexpress HER2 don't receive benefit from such treatment.Trastuzumab can be highly effective, but 70% of HER2 positive tumorsdon't respond to treatment and others may eventually develop resistance.Trastuzumab can also cause heart damage Radiation therapy can be usedbut also causes heart problems. The methods of the invention can be usedto identify treatment regimens including the above standard drugs andnon-standard drugs for treatment of breast cancer.

The subject methods can be used to identify a candidate treatment for asubject suffering from breast cancer comprising a triple-receptornegative breast cancer. Triple negative breast cancer includes breastcancer that expresses little to no ER or PR, and does not exhibitoverexpression and/or gene amplification of HER2/neu. See, e.g., DawoodS, Broglio K, Esteva F J, Yang W, Kau S W, Islam R, Albarracin C, Yu TK, Green M, Hortobagyi G N, Gonzalez-Angulo A M. Survival among womenwith triple receptor-negative breast cancer and brain metastases. AnnOncol. 2009 April; 20(4):621-7. Epub 2009 Jan. 15. Illustrative diagramsfor identifying candidate treatments according to the invention areshown in FIGS. 42 and 43. FIG. 42 shows a flow diagram and FIG. 43 showsbiomarkers that can be assessed. The subject may have metastatic breastcancer and completed a first, second, or third line of therapy. IHCprofiling can be conducted on a sample from the subject to determine thepresence or level of one or more of: AR, CK5/6, CK14, CK17, ER, HER2,Ki67, MRP1, P53, PGP, PR, SPARC and TS. In some embodiments, IHC isconducted on all of these biomarkers. The IHC analysis can be combinedwith microarray analysis and/or assessment of HER2 by fluorescentin-situ hybridization (FISH). The IHC analysis can determine thepresence or level of additional biomarkers, such as one or more of:BCRP, c-KIT, ERCC1, MGMT, PDGFR, PTEN, RRM1, and TOP2A. In someembodiments, IHC is conducted on all of these additional biomarkers. Thesubject may have completed a fourth line of therapy or beyond. Based onresults for one or more of the foregoing (i.e., IHC, FISH, sequencing,microarray), a treatment or therapy is selected. Based on the analysis,a likelihood of clinical benefit or lack of clinical benefit of aparticular candidate treatment is determined. Illustrative treatmentsinclude without limitation an anthracycline, taxane, platinum analog,anti-neoplastic agent, camptothecin, pyrimidine analog, vinca alkaloid,gonatropin releasing hormone analog, anti-androgen, or any combinationthereof, such as doxorubicin, liposomal doxorubicin, epirubicin,paclitaxel, docetaxel, nab-paclitaxel, carboplatin, cisplatin,gemcitabine, irinotecan, capecitabine, 5-fluorouracil, vinblastine,vinorelbine, goserelin, leuprolide, bicalutamide, or flutamide.

The subject methods can be used to identify a candidate treatment for asubject suffering from breast cancer that is hormone-receptor-positiveand HER2 negative (ER+ and/or PR+, and HER2−). Illustrative diagrams foridentifying candidate treatments according to the invention are shown inFIGS. 42 and 43. FIG. 42 shows a flow diagram and FIG. 43 showsbiomarkers that can be assessed. The subject's HER2 status may havechanged. The subject may have metastatic breast cancer and completed afirst, second, or third line of therapy. IHC profiling can be conductedon a sample from the subject to determine the presence or level of oneor more of: CAV-1, c-KIT, CYCLIN D1, EGFR, ER, HER2, Ki67, p53, PR,PDGFR, PGP, PTEN and TS. In some embodiments, IHC is conducted on all ofthese biomarkers. The IHC analysis can be combined with microarrayanalysis and/or assessment of HER2, cMYC, or both, by fluorescentin-situ hybridization (FISH). The IHC analysis can determine thepresence or level of additional biomarkers, such as one or more of: AR,ERCC1, MGMT, MRP1, RRM1, SPARC, TOP1, and TOP2A. In some embodiments,INC is conducted on all of these additional biomarkers. The subject mayhave completed a fourth line of therapy or beyond. Based on results forone or more of the foregoing (i.e., IHC, FISH, sequencing, microarray),a treatment or therapy is selected. Based on the analysis, a likelihoodof clinical benefit or lack of clinical benefit of a particularcandidate treatment is determined. Illustrative treatments includewithout limitation a monoclonal antibody, protein kinase inhibitor,anthracycline, taxane, platinum analog, anti-neoplastic agent,camptothecin, anti-estrogen therapy, armatase inhibitor, pyrimidineanalogue, vinca alkaloid, gonatropin releasing hormone analogue,anti-androgen, folic acid analog, selective estrogen receptor modulator,or any combination thereof, such as trastuzumab, lapatinib, doxorubicin,liposomal doxorubicin, epirubicin, paclitaxel, docetaxel,nab-paclitaxel, carboplatin, cisplatin, gemcitabine, irinotecan,fulvestrant, anastrozole, exemestane, letrozole, capecitabine,5-fluorouracil, vinblastine, vinorelbine, leuprolide, bicalutamide,flutamide, goserelin, methotrexate, tamoxifen, or toremifene.

The subject methods can be used to identify a candidate treatment for asubject suffering from breast cancer that is HER2 positive (HER2+).Illustrative diagrams for identifying candidate treatments according tothe invention are shown in FIGS. 42 and 43. FIG. 42 shows a flow diagramand FIG. 43 shows biomarkers that can be assessed. The subject's HER2status may have changed or has progressed on trastuzumab. The subjectmay have metastatic breast cancer and completed a first, second, orthird line of therapy. IHC profiling can be conducted on a sample fromthe subject to determine the presence or level of one or more of:E-cadherin, ER, HER2, Ki67, MRP1, p53, p95, PGP, PR, PTEN, TLE3 and TS.In some embodiments, INC is conducted on all of these biomarkers. TheIHC analysis can be combined with microarray analysis, fluorescentin-situ hybridization (FISH) assessment of HER2, cMYC, TOP2A, or acombination, and sequencing of PIK3CA. The IHC analysis can determinethe presence or level of additional biomarkers, such as one or more of:AR, BCRP, c-KIT, ERCC1, MGMT, PDGFR, RRM1, SPARC, TOP1, and TOP2A. Insome embodiments, IHC is conducted on all of these additionalbiomarkers. In some embodiments, the subject has completed a fourth lineof therapy or beyond. Based on results for one or more of the foregoing(i.e., IHC, FISH, sequencing, microarray), a treatment or therapy isselected. Based on the analysis, a likelihood of clinical benefit orlack of clinical benefit of a particular candidate treatment isdetermined. Illustrative treatments include without limitation amonoclonal antibody, protein kinase inhibitor, anthracycline, taxane,platinum analog, anti-neoplastic agent, camptothecin, anti-estrogentherapy, armatase inhibitor, pyrimidine analogue, vinca alkaloid,gonatropin releasing hormone analogue, anti-androgen, folic acid analog,selective estrogen receptor modulator, or any combination thereof, suchas trastuzumab, lapatinib, doxorubicin, liposomal doxorubicin,epirubicin, paclitaxel, docetaxel, nab-paclitaxel, carboplatin,cisplatin, gemcitabine, irinotecan, fulvestrant, anastrozole,exemestane, letrozole, capecitabine, 5-fluorouracil, vinblastine,vinorelbine, leuprolide, bicalutamide, flutamide, goserelin,methotrexate, tamoxifen, or toremifene.

In one aspect, the invention provides a method for identifying atherapeutic for an individual with breast cancer comprising: a)determining an expression level or a mutation of a gene from abiological sample of the individual, wherein the gene is selected fromthe group consisting of: ER, PR, HER2, Ki-67 and P53; and b) identifyinga therapeutic for treating the individual based on a change inexpression or a mutation as compared to a reference. The expressionlevel or mutation can be determined by, e.g., IHC, FISH, microarray,sequencing, real-time PCR or other methods as disclosed herein. Theresults can be used to subtype the breast cancer, e.g., according toreceptor status or drug resistance status. In some embodiments, thebreast cancer comprises an Invasive Breast Cancer. In some embodiments,the breast cancer is Her-2 positive. Her-2 expression can be determinedby FISH and/or IHC. In some embodiments, the breast cancer comprises atriple negative breast cancer. The cancer can also be metastatic. Insome embodiments, the breast cancer is negative for at least one of ER,PR, or Her2. In some embodiments, the breast cancer is negative for atleast two of ER, PR, or Her2. In other embodiments, the breast cancer isnegative for at least one of ER, PR, or Her-2, and positive for at leastone of ER, PR, or Her2. In some embodiments, the breast cancer isnegative for at least two of ER, PR, or Her2, e.g. ER-negative,PR-negative, and Her-2 positive; or ER-positive, PR-negative, and Her2negative; or ER-negative, PR-positive, and Her2 negative. In oneembodiment, the breast cancer is an ER and/or PR+, HER2− breast cancer.The subtype of the breast cancer can be further used to identify orrefine a therapeutic.

In one embodiment, the breast cancer is Her-2 positive. About 20-30% ofbreast cancers are HER2 positive. In HER2+ breast cancer, the cancercells have an abnormally high number of HER2 genes per cell. When thishappens, an abundance of HER2 protein appears on the surface of thesecancer cells. Of these, about 30% respond to trastuzumab therapy. Theresponse may be dependent on loss of PTEN, PI3 Kinase mutations, p95HER2expression, and/or IGF-1R expression. p95HER2 refers to a truncated formof the HER2 receptor. In one embodiment, HER-2 status is determined byFISH and/or IHC. In some embodiments, the invention provides a method ofdetermining a therapeutic treatment for an individual having HER-2positive breast cancer comprising: a) determining an expression level ofa gene and/or a mutation in a gene from a biological sample of theindividual, wherein the gene is selected from the group consisting of:HER2, PTEN, PI-3 kinase, IGF-IR and p95HER2; and b) identifying atherapeutic based on the mutation or wherein the gene exhibits a changein expression as compared to a reference. Some of the individuals willrespond to lapatinib or trastuzumab. In some embodiments, loss of PTEN,mutation in PI-3 Kinase, over expression of IGF-1R or over expressionp95HER2 indicates decreased probability of response to trastuzumab andcan favor treatment with lapatinib. In some embodiments, the panel foridentifying a therapeutic for an individual having HER2 breast cancercomprises analysis of expression and/or mutation of HER2, PTEN, IGF-1Rand p95HER2, PI-3 Kinase, or a combination thereof. In some embodiments,the panel comprises TOP2A, PGP, MRP1, TS, ERCC1, BCRP, RRM1, TOPOI,TOPOII, TLE3 (for taxanes), C-MYC, TOP2, P95, PTEN, E-Cad, HER2, PI3K ora combination thereof. For example, BCRP, ERCC1, MRP1, p95, PGP, RRM1,TLE3, TopoI, TopoII, TS, PTEN and E-cad can be assayed by IHC, HER2,cMYC and TOP2A can be assayed by FISH, and PI3K can be assayed bysequencing. The panels can be used to identify therapeutics for relapsedor refractive cancers.

In one embodiment, the breast cancer is a triple negative breast cancer.Triple negative breast cancer, which refers to cancers that are estrogenreceptor (ER) negative, progesterone receptor (PR) negative, and humanepidermal growth factor receptor 2 (Her-2) negative, compriseapproximately 15% of all breast cancers and have an aggressive clinicalcourse with high rates of local and systemic relapse. The clinicalcourse reflects the biology of the tumor as well as the absence ofconventional targets for treatment such as hormonal therapy for ER or PRpositive patients and trastuzumab for Her-2 over-expressing tumors.Despite the availability of antimetabolites such as gemcitabine andplatinum complex agents such as carboplatin, there is no acceptedstandard of care for ER negative breast cancer. In particular, triplenegative metastatic breast cancer is refractory to standard treatmentsand is refractory to serum estrogen receptor modulator (SEAM)chemotherapy.

DNA repair deficits can be a characteristic of triple negative cancers.Such cancers frequently harbor defects in DNA double-strand break repairthrough homologous recombination (UR), such as BRCA1 dysfunction(Rottenberg S, et. al. Proc Natl Acad Sci USA. 2008 Nov. 4;105(44):17079-84). These tumors exhibit more DNA copy alterations andloss of heterozygosity than other breast cancers, features suggestive ofgenomic instability. Furthermore, sporadic triple negative tumors sharephenotypic and cytogenetic features with familial BRCA1 associatedcancer and correlate with BRCA1 cancers using microarray RNA expressiondata. BRCA1 mutant tumors are thought to be deficient in DNA repair,particularly homologous recombination, and these similarities maysuggest that a similar DNA repair deficiency may play a role in triplenegative tumors.

In some embodiments, the invention provides a method of determining atherapeutic treatment for an individual having a triple negative breastcancer breast cancer comprising: a) determining an expression level of agene and/or a mutation in a gene from a biological sample of theindividual, wherein the gene is selected from the group consisting of:AR, KRAS, BRCA1, PARP-1, SPARC, CK 5/6, CK14, CK17, TOP2A, PGP, MRP1,TS, ERCC1, BCRP, RRM1, TOPOI, TOPOII, TLE3; and b) identifying atherapeutic based on the mutation or wherein the gene exhibits a changein expression as compared to a reference. In some embodiments, AR, KRAS,BRCA1, PARP-1, SPARC, CK 5/6, CK14, CK17, TOP2A, PGP, MRP1, TS, ERCC1,BCRP, RRM1, TOPOI, TOPOII TLE3 are assayed using IHC. In someembodiments, KRAS is assayed by sequencing. The panel can be used toidentifying therapeutics for relapsed or refractive cancers.

In some embodiments, the breast cancer comprises Ductal Carcinoma inSitu (DCIS). In one aspect, the invention provides a method foridentifying a therapeutic for an individual with DCIS comprising: a)determining an expression level of a gene from a biological sample ofthe individual, wherein the gene is selected from the group consistingof: ER, PR HER2, Ki-67, P53, BCL2 and E-Cadherin; and b) identifying atherapeutic that the individual has not previously been treated for thecondition, when the gene exhibits a change in expression as compared toa reference. The expression levels can be determined by, e.g., IHC,FISH, microarray, sequencing, real-time PCR or other methods asdisclosed herein. A therapeutic can be chosen based on the expression ofthe gene or of a mutation thereof.

In an aspect, the invention provides a method for identifying atherapeutic for an individual having breast cancer comprising: (a)determining an expression level of a gene and/or a mutation in a genefrom a biological sample of the individual, wherein the gene is selectedfrom the group consisting of: SPARC, TOP2A, TOTO1, PGP, BCRP, MRP1,PTEN, TS, ERCC1, RRM1, MGMT, c-kit, PDGFR, AR, EGFR, KRAS, BRAF, p95 orPI3K; and (b) identifying a therapeutic for the individual when the geneexhibits a change in expression as compared to a reference. In someembodiments, the individual has refractive breast cancer or hasrelapsed. The cancer can be metastatic. The expression and/or themutation can be determined using IHC, FISH, microarray, sequencing,real-time PCR or other methods as disclosed herein.

In a related aspect, the invention provides a method of identifying acandidate treatment for a subject in need thereof by using molecularprofiling of sets of known biomarkers. For example, the method canidentify a chemotherapeutic agent for an individual with a cancer. Themethod comprises: obtaining a sample from the subject; performing animmunohistochemistry (IHC) analysis on the sample to determine an IHCexpression profile on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore, of: AR, c-Kit, CAV-1, CK 5/6, CK14, CK17, ECAD, ER, Her2/Neu,Ki67, MRP1, P53, P95, PDGFR, PGP, PR, PTEN, SPARC (using a monoclonaland/or polyclonal antibody), TLE3, TOP2A and TS; performing a microarrayanalysis on the sample to determine a microarray expression profile onone or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, of: ABCC1, ABCG2,ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK,DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3,ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1,IL2RA, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR,PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2,RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1,TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70;performing a fluorescent in-situ hybridization (FISH) analysis on thesample to determine a FISH mutation profile on at least HER2. If thecancer is a HER2+ breast cancer, the method further comprises:performing FISH analysis on the sample to determine a FISH mutationprofile for cMYC and TOP2A; and performing DNA sequencing on the sampleto determine a sequencing mutation profile on at least PI3K (PIK3CA). Ifthe cancer is (ER+ or PR+) and HER2− breast cancer, the method furthercomprises: performing IHC analysis on the sample to determine an IHCexpression profile on one or more of Cyclin D1 and EGFR; and performingFISH analysis on the sample to determine a FISH mutation profile forcMYC. If the cancer comprises. 1) triple negative (i.e., ER−, PR− andHER2−) breast cancer, 2) HER2+ breast cancer, or 3) (ER+ or PR+) andHER2− breast cancer, and the cancer is fourth line, metastatic orbeyond, or has the therapeutic history is not known, the method furthercomprises: performing IHC analysis on the sample to determine an IHCexpression profile on one or more of BCRP, ERCC1, MGMT, RRM1 and TOPO1;and performing FISH analysis on the sample to determine a FISH mutationprofile for EGFR. The molecular profiling according to the method isillustrated in FIGS. 42 and 43. Once the molecular profiling isperformed, the method further comprises comparing the IHC expressionprofile, microarray expression profile, FISH mutation profile andsequencing mutation profile against a rules database, wherein the rulesdatabase comprises a mapping of treatments whose biological activity isknown against diseased cells that: i) overexpress or underexpress one ormore proteins included in the INC expression profile; ii) overexpress orunderexpress one or more genes included in the microarray expressionprofile; iii) have zero or more mutations in one or more genes includedin the FISH mutation profile; and/or iv) have zero or more mutations inone or more genes included in the sequencing mutation profile; andidentifying the treatment if the comparison against the rules databaseindicates that the treatment should have biological activity against thecancer; and the comparison against the rules database does notcontraindicate the treatment for treating the cancer. In someembodiments, the IHC expression profiling is performed on at least 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the gene products above. Insome embodiments, the microarray expression profiling is performed on atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the genes listedabove. In some embodiments, IHC is performed on 100% of the geneproducts indicated above. The microarray expression profiling can alsobe performed on 100% of the genes indicated above. The molecularprofiling steps can be performed in any order. In some embodiments, notall of the molecular profiling steps are performed. As a non-limitingexample, microarray analysis is not performed if the sample quality doesnot meet a threshold value, as described herein. In some embodiments,the biological material is mRNA and the quality control test comprises aA260/A280 ratio and/or a Ct value of RT-PCR using a housekeeping gene,e.g., RPL13a. In embodiments, the mRNA does not pass the quality controltest if the A260/A280 ratio <1.5 or the RPL13a Ct value is >30. In thatcase, microarray analysis may not be performed. Alternately, microarrayresults may be attenuated, e.g., given a lower priority as compared tothe results of other molecular profiling techniques.

Breast Cancer Panels

As described herein, biomarkers can be assessed to indicate candidatetherapeutics for treatment of breast cancer. The candidate therapeuticscan be selected based on the molecular profiling panels presented inthis Section.

Approximately 42% to 59% of breast cancers are of the hormone receptorpositive A subtype, 6% to 19% are hormone receptor positive B. (KomenFoundation. Molecular Subtypes of Breast Cancer.ww5.komen.org/content.aspx?id=5372 Last accessed May 17, 2010). Hormonereceptor positive A tumors tend to have the best prognosis, with highsurvival rates and low recurrence rates. Hormone receptor positive Bpatients have a lower survival rate compared with hormone receptorpositive A patients.

Molecular profiling can help determine the status of a subject's hormonereceptor positive, HER-2 negative breast cancer and to deliver anevidence-based report with individualized therapeutic guidance.Biomarker data derived from the tests listed in Table 13 can be used tomake informed treatment decisions for hormone receptor positive, HER-2negative cancer patients, including without limitation those who aremetastatic and have completed 3^(rd) line therapy, or are metastatic andtheir HER-2 status has changed, or who have unique circumstances thatcreate questions for their therapeutic management, or have exhaustedstandard of care therapies.

Examples of drug therapies that may be associated with clinical benefitor lack of clinical benefit based on biomarker status include MonoclonalAntibody (trastuzumab), Protein Kinase Inhibitor (lapatinib),Anthracyclines (doxorubicin, liposomal doxorubicin, epirubicin), Taxanes(paclitaxel, docetaxel, nab-paclitaxel), Platinum Analogs (carboplatin,cisplatin), Anti-Neoplastic Agent (gemcitabine), Camptothecin(irinotecan), Anti-Estrogen Therapy (fulvestrant), Armatase Inhibitors(anastrozole, exemestane, letrozole), Pyrimidine Analogues(capecitabine, 5-fluorouracil), Vinca Alkaloids (vinblastine,vinorelbine), Gonatropin Releasing Hormone Analogues (goserelin,leuprolide), Anti-Androgens (bicalutamide, flutamide, goserelin), FolicAcid Analogue (methotrexate), Selective Estrogen Receptor Modulators(tamoxifen, toremifene).

TABLE 13 Molecular Profiling for Hormone Receptor Positive and HER2Negative Breast Cancer: Biomarkers Assessed Third line metastatic orprior Fourth line metastatic or beyond IHC IHC CAV-1 P53 AR HER2 PTENc-KIT P95 BCRP Ki67 RRM1 CYCLIN D1 PR CAV-1 MGMT SPARC EGFR PDGFR CYCLIND1 MRP1 Mono ER PGP c-KIT P53 SPARC HER2 PTEN EGFR P95 Poly Ki67 TS ERPDGFR TOPO1 ERCC1 PGP TOP2A PR TS FISH FISH   HER2 cMYC   HER2 cMYCMutation Analysis Mutation Analysis   NA   NA DNA Microarray DNAMicroarray   Whole genome expression   Whole genome expression array  array

Approximately 25% of breast cancers overexpress HER-2. These tumors tendto grow faster and are generally more likely to recur than tumors thatdo not overproduce HER-2. (National Cancer Institute. Breast CancerTreatment (PDQ®), available atwww.cancer.gov/cancertopics/pdq/treatment/breast/HealthProfessional/page8)A challenge for treating physicians is properly selecting the order ofavailable treatment agents when the patient progresses beyond standardof care.

Molecular profiling can help determine the status of a subject's HER-2positive breast cancer and to deliver an evidence-based report withindividualized therapeutic guidance. Biomarker data derived from thetests listed in Table 14 can be used to make informed treatmentdecisions for HER-2 positive breast cancer patients, including withoutlimitation those who have progressed on trastuzumab, or are metastaticand have completed 3^(rd) line therapy, or are metastatic and theirHER-2 status has changed, or have unique circumstances that createquestions for their therapeutic management, or have exhausted standardof care therapies.

Examples of drug therapies that may be associated with clinical benefitor lack of clinical benefit based on biomarker status include MonoclonalAntibody (trastuzumab), Protein Kinase Inhibitor (lapatinib),Anthracyclines (doxorubicin, liposomal doxorubicin, epirubicin), Taxanes(paclitaxel, docetaxel, nab-paclitaxel), Platinum Analogs (carboplatin,cisplatin), Anti-Neoplastic Agent (gemcitabine), Camptothecin(irinotecan), Anti-Estrogen Therapy (fulvestrant), Armatase Inhibitors(anastrozole, exemestane, letrozole), Pyrimidine Analogues(capecitabine, 5-fluorouracil), Vinca Alkaloids (vinblastine,vinorelbine), Gonatropin Releasing Hormone Analogues (goserelin,leuprolide), Anti-Androgens (bicalutamide, flutamide, goserelin), FolicAcid Analogue (methotrexate), Selective Estrogen Receptor Modulators(tamoxifen, toremifene).

TABLE 14 Molecular Profiling for HER2 Positive Breast Cancer: BiomarkersAssessed Third line Fourth line metastatic or prior metastatic or beyondIHC IHC E-cadherin P95 AR HER2 PDGFR SPARC Mono ER PGP BCRP Ki67 PGPSPARC Poly HER2 PR c-KIT MGMT PR TLE3 Ki67 PTEN E-cadherin MRP1 PTENTOPO1 MRP1 TLE3 ER P53 RRM1 TOP2A P53 TS ERCC1 P95 TS FISH FISH   HERZcMYC   HER2 cMYC TOP2A TOP2A Mutation Analysis Mutation Analysis  PIK3CA   PIK3CA DNA Microarray DNA Microarray   Whole genome   Wholegenome expression array   expression array

Approximately 10% to 15% of breast cancers are known to be“triple-receptor-negative.” (Dawood S, Broglio K, Esteva F J, Yang W,Kau S W, Islam R, Albarracin C, Yu T K, Green M, Hortobagyi G N,Gonzalez-Angulo A M. Survival among women with triple receptor-negativebreast cancer and brain metastases. Ann Oncol. 2009 Apm20(4):621-7. Epub2009 Jan. 15.) Patients with triple negative breast cancer are morelikely to relapse during the first 3 years following therapy. (Bauer KR, Brown M, Cress R D, Parise C A, Caggiano V. Descriptive analysis ofestrogen receptor (ER)-negative, progesterone receptor (PR)-negative,and HER2-negative invasive breast cancer, the so-called triple-negativephenotype: a population-based study from the California cancer Registry.Cancer. 2007 May 1; 109(9):1721-8.) The relative survival for all womenwith triple-negative tumors is 77% at 5 years, compared with 93% forother breast cancers. (Bauer K R, Brown M, Cress R D, Parise C A,Caggiano V. Descriptive analysis of estrogen receptor (ER)-negative,progesterone receptor (PR)-negative, and HER2-negative invasive breastcancer, the so-called triple-negative phenotype: a population-basedstudy from the California cancer Registry. Cancer. 2007 May 1;109(9):1721-8.)

Molecular profiling can help determine the status of a subject'striple-negative breast cancer and to deliver an evidence-based reportwith individualized therapeutic guidance. Biomarker data derived fromthe tests listed in Table 15 can be used to make informed treatmentdecisions for triple-negative breast cancer patients, including withoutlimitation those who are basal type and/or triple negative, or aremetastatic and have completed 3^(rd) line therapy, or have uniquecircumstances that create questions for their therapeutic management, orhave exhausted standard of care therapies.

Examples of drug therapies that may be associated with clinical benefitor lack of clinical benefit based on biomarker status includeAnthracyclines (doxorubicin, liposomal doxorubicin, epirubicin), Taxanes(paclitaxel, docetaxel, nab-paclitaxel), Platinum Analogs (carboplatin,cisplatin), Anti-Neoplastic Agent (gemcitabine), Camptothecin(irinotecan), Pyrimidine Analogues (capecitabine, 5-fluorouracil), VincaAlkaloids (vinblastine, vinorelbine), Gonatropin Releasing HormoneAnalogues (goserelin, leuprolide), Anti-Androgens (bicalutamide,flutamide, goserelin).

TABLE 15 Molecular Profiling for Triple-Negative Breast Cancer:Biomarkers Assessed Third line Fourth line metastatic or priormetastatic or beyond IHC IHC AR Ki67 AR Ki67 RRM1 CK 5/6 MRP1 BCRP MGMTSPARC Mono CK 14 P53 CK 5/6 MRP1 SPARC Poly CK 17 P95 CK 14 P53 TLE3 ERPGP CK 17 P95 TOPO1 HER2 PR c-KIT PDGFR TOP2A SPARC Mono ER PGP TS SPARCPoly ERCC1 PR TS HER2 PTEN FISH FISH   HER2   HER2 Mutation AnalysisMutation Analysis   NA   NA DNA Microarray DNA Microarray   Whole genomeexpression   Whole genome expression array   array

Prognostics

In another aspect, the invention provides a method of providing aprognosis for a cancer. The method comprises performing molecularprofiling on the sample as described herein and providing a prognosisbased on the molecular profiling results. Accordingly, molecularprofiling can be used to simultaneously identify a candidate therapeuticand provide a prognosis. In an embodiment, the method for prognosing acancer in an individual comprises. (a) determining a level of a gene orgene product and/or a mutation in a gene from a biological sample of theindividual, wherein the gene is selected from the group of genes listedin andy of Tables 11-15; and (b) prognosing the cancer based whether thegene is up or down regulated in the cancer as compared to a control.Table 16 indicates whether the differential regulation of the gene, orgene product thereof, as compared to the control indicates a goodprognosis or bad prognosis. In the table, presence and absence alsorefer to overexpression and underexpression, respectively, as comparedto the control. Any appropriate control can be used. In embodiments, thecontrol comprises a non-diseased sample from the individual or fromanother individual. The method can be applied to the various cancersdescribed herein. For example, the cancer assessed can be a breastcancer. In some embodiments, the individual has refractive cancer or hasrelapsed. The cancer can be metastatic. The expression and/or themutation can be determined using IHC, FISH, microarray, sequencing,real-time PCR or other molecular profiling methods as disclosed herein.In an embodiment, IHC is used to determine the expression of the proteincomprising the gene product. In another embodiment, DNA microarrayanalysis is used. The method can be performed using the same molecularprofiling results as the theranostic methods of the invention. In thismanner, the invention provides a method for analyzing a cancer tosimultaneously identify a candidate therapeutic and provide a prognosis.

TABLE 16 Prognostic Markers Biomarker Summary Caveolin 1 Presence ofCav-1 indicates good prognosis. Caveolin 1 Absence of Cav-1 indicatesbad prognosis. CK5/6 Presence of CK5/6 indicates bad prognosis. CK5/6Absence of CK5/6 indicates good prognosis. CK14 Presence of CK14indicates bad prognosis. CK14 Absence of CK14 indicates good prognosis.CK17 Presence of CK17 indicates bad prognosis. CK17 Absence of CK17indicates good prognosis. C-kit Presence of c-kit indicates badprognosis. C-kit Absence of c-kit indicates good prognosis. c-mycAmplification of c-myc indicates bad prognosis. c-myc Non-amplificationof c-myc indicates good prognosis. Cyclin D1 Presence of Cyclin D1indicates bad prognosis. Cyclin D1 Absence of Cyclin D1 indicates goodprognosis. E-cadherin Presence of E-cadherin indicates good prognosis.E-cadherin Absence of E-cadherin indicates bad prognosis. EGFR Presenceof EGFR indicates bad prognosis. EGFR Absence of EGFR indicates goodprognosis. P53 Presence of P53 indicates good prognosis. P53 Absence ofP53 indicates bad prognosis. PDGFR Presence of PDGFR indicates badprognosis. PDGFR Absence of PDGFR indicates good prognosis.

In some embodiments, the prognostic markers are themselves associatedwith a candidate therapeutic. In other embodiments, prognostic markersare assessed simultaneously with the markers associated with candidatetherapeutics as part of the molecular profile. Even in this latter casethe markers can inform the selection of a candidate therapeutic. Forexample, a marker status indicating a worse prognosis can indicate aneed for a more aggressive treatment regimen. Likewise, a marker statusindicating a good prognosis can indicate a need for a less aggressivetreatment regimen.

Cancer of Unknown Primary

Molecular profiling can be used to determine a treatment for a cancerregardless of its origin, making this approach particularly attractivefor treating a CUP patient. As described herein, the type of tumor canalso provide informative information to guide treatment selection. See,e.g., FIG. 46A-B. Molecular profiling can be used to identify a profileto identify a tumor's origin. In an aspect, the invention provides amethod of identifying a candidate treatment by performing molecularprofiling on a CUP sample, thereby identifying the candidate treatmentand/or the origin of the tumor. In one embodiment, \ expression analysisis performed on the CUP sample, and the expression profile is used toidentify the tumor origin. The origin is then used to select furthermolecular profiling tests to be performed on the sample, e.g., IHC, FISHand/or mutational analysis. The combined results of the expressionanalysis, IHC, FISH and/or mutational analysis are used to select acandidate treatment. Expression analysis can comprise DNA microarray,PCR-based arrays, protein array, mass spectroscopy, or other techniquesuseful for determining expression of a plurality of genes and/or geneproducts.

In an embodiment, expression profiling is used to identify a molecularprofile to differentiate breast cancer from other types of cancer. Thepanel of genes to be assessed can comprise one or more of AK5.2,ATP6V1B1, CRABP1, DST.3, GATA3, KRT81, ELF5, LALBA, OXTR, RASL10A,SERHL, TFAP2A.1, TFAP2A.3, TFAP2C and VTCN1. One useful subset of thesegenes for distinguishing a breast cancer comprises AK5.2, ATP6V1B1,and/or CRABP1. Another useful subset of these genes for distinguishing abreast cancer comprises DST.3, GATA3, and/or KRT81. Still another usefulsubset subset of these genes for distinguishing a breast cancercomprises AK5.2, ATP6V1B1, CRABP1, DST.3, GATA3, KRT81, ELF5, LALBA,OXTR, RASL10A, SERHL, TFAP2A.1, TFAP2A.3, TFAP2C and VTCN. One of skillwill appreciate that molecular profiles for other tumor origins can beused to distinguish other tumor types. As described herein, themolecular profiles and/or origins can be used to guide treatmentselection as desired. See, e.g., FIG. 46A-B.

EXAMPLES Example 1 IHC and Microarray Testing of Over 500 Patients

The data reflected in the table depicted in FIGS. 26A-H and FIGS.27A-27H relates to 544 patients whose diseased tissue samples underwentIHC testing (FIG. 26) and 540 patients whose diseased tissue samplesunderwent gene microarray testing (FIG. 27) in accordance with IHC andmicroarray testing as previously described above. The patients were allin advanced stages of disease.

The data show biomarker patterns or biomarker signature sets in a numberof tumor types, diseased tissue types, or diseased cells includingadipose, adrenal cortex, adrenal gland, adrenal gland—medulla, appendix,bladder, blood vessel, bone, bone cartilage, brain, breast, cartilage,cervix, colon, colon sigmoid, dendritic cells, skeletal muscle,endometrium, esophagus, fallopian tube, fibroblast, gallbladder, kidney,larynx, liver, lung, lymph node, melanocytes, mesothelial lining,myoepithelial cells, osteoblasts, ovary, pancreas, parotid, prostate,salivary gland, sinus tissue, skeletal muscle, skin, small intestine,smooth muscle, stomach, synovium, joint lining tissue, tendon, testis,thymus, thyroid, uterus, and uterus corpus.

In 99 individuals with advanced breast cancer, immunohistochemistryanalysis of 20 gene expressed proteins (FIG. 26B) showed that the geneexpressed proteins analyzed were overexpressed a total of 367 times andthat 16.35% of that total overexpression was attributable to HSP90overexpression followed by 12.53% of the overexpression beingattributable to TOP2A overexpression and 11.17% of the overexpressionbeing attributable to SPARC. In addition, 9.81% of the overexpressionwas attributable to androgen receptor overexpression, 9.54% of theoverexpression was attributable to PDGFR overexpression, and 9.26% ofthe overexpression was attributable to c-kit overexpression.

Accordingly, a biomarker pattern or biomarker signature set can beidentified for advanced stage breast cancer and a therapeutic agent ortherapeutic protocol can be identified which is capable of interactingwith the biomarker pattern or signature set.

Another biomarker pattern or biomarker signature set for advanced stagebreast cancer is shown from the microarray data in the table representedby FIGS. 27A-H. For example, in 100 individuals with advanced breastcancer (FIG. 27B), gene microarray analysis of 64 genes showed that thegenes analyzed exhibited a change in expression a total of 1,158 timesand that 6.39% of that total change in expression was attributable toSSTR3 change in expression followed by 5.79% of the change in expressionbeing attributable to VDR change in expression and 5.35% of the changein expression being attributable to BRCA2 change in expression.Accordingly, another biomarker pattern or biomarker signature set can beidentified for advanced stage breast cancer and another therapeuticagent or therapeutic protocol can be identified which is capable ofinteracting with this biomarker pattern or signature set.

Example 2 IHC Testing of Over 1300 Patients

FIGS. 28A through 28O represent a table that shows the frequency of asignificant change in expression of certain gene expressed proteins bytumor type, i.e. the number of times that a gene expressed protein wasflagged as a target by tumor type as being significantly overexpressedby immunohistochemistry analysis. The table also identifies the totalnumber of times an overexpression of any gene expressed protein occurredin a particular tumor type using immunohistochemistry.

The data reflected in the table depicted in FIGS. 28A through 28Orelates to 1392 patients whose diseased tissue underwent IHC testing inaccordance with IHC testing as previously described above. The patientswere all in advanced stages of disease.

The data show biomarker patterns or biomarker signature sets in a numberof tumor types, diseased tissue types, or diseased cells includingaccessory, sinuses, middle and inner ear, adrenal glands, appendix,hematopoietic system, bones and joints, spinal cord, breast, cerebellum,cervix uteri, connective and soft tissue, corpus uteri, esophagus, eye,nose, eyeball, fallopian tube, extrahepatic bile ducts, other mouth,intrahepatic bile ducts, kidney, appendix-colon, larynx, lip, liver,lung and bronchus, lymph nodes, cerebral, spinal, nasal cartilage, excl.retina, eye, nos, oropharynx, other endocrine glands, other femalegenital, ovary, pancreas, penis and scrotum, pituitary gland, pleura,prostate gland, rectum renal pelvis, ureter, peritonem, salivary gland,skin, small intestine, stomach, testis, thymus, thyroid gland, tongue,unknown, urinary bladder, uterus, nos, vagina & labia, and vulva, nos.

In 254 individuals with advanced breast cancer, immunohistochemistryanalysis of 19 gene expressed proteins (FIG. 28C) showed that the geneexpressed proteins analyzed were overexpressed a total of 767 times andthat 13.43% of that total overexpression was attributable to SPARCoverexpression followed by 12.26% of the overexpression beingattributable to c-kit overexpression and 11.47% of the overexpressionbeing attributable to EGFR. In addition, 11.34% of the overexpressionwas attributable to androgen receptor overexpression, 11.08% of theoverexpression was attributable to HSP90 overexpression, and 10.43% ofthe overexpression was attributable to PDGFR overexpression.Accordingly, a biomarker pattern or biomarker signature set can beidentified for advanced stage breast cancer and a therapeutic agent ortherapeutic protocol can be identified which is capable of interactingwith the biomarker pattern or signature set.

FIG. 29 depicts a table showing biomarkers (gene expressed proteins)tagged as targets in order of frequency in all tissues that were IHCtested. Immunohistochemistry of the 19 gene expressed proteins showedthat the 19 gene expressed proteins were tagged 3878 times as targets inthe various tissues tested and that EGFR was the gene expressed proteinthat was overexpressed the most frequently followed by SPARC.

Example 3 Microarray Testing of Over 300 Patients

FIGS. 30A through 30O represent a table that shows the frequency of asignificant change in expression of certain genes by tumor type, i.e.the number of times that a gene was flagged as a target by tumor type asbeing significantly overexpressed or underexpressed by microarrayanalysis. The table also identifies the total number of times anoverexpression or underexpression of any gene occurred in a particulartumor type using gene microarray analysis.

The data reflected in the table depicted in FIGS. 30A through 30Orelates to 379 patients whose diseased tissue underwent gene microarraytesting in accordance microarray testing as previously described above.The patients were all in advanced stages of disease. The data showbiomarker patterns or biomarker signature sets in a number of tumortypes, diseased tissue types, or diseased cells including accessory,sinuses, middle and inner ear, adrenal glands, anal canal and anus,appendix, blood, bone marrow & hematopoietic sys, bones and joints,brain & cranial nerves and spinal cord (excl. ventricle & cerebellum),breast, cerebellum, cervix uteri, connective & soft tissue, corpusuteri, esophagus, eye, nos, eyeball, fallopian tube, gallbladder 7extrahepatic bile ducts, gum, floor of mouth & other mouth, intrahepaticbile ducts, kidney, large intestine (excl. appendix-colon), larynx, lip,liver, lung & bronchus, lymph nodes, meninges (cerebral, spinal), nasalcavity (including nasal cartilage), orbit & lacrimal gland (excl.retina, eye, nos), oropharynx, other endocrine glands, other fenalegenital, ovary, pancreas, penis & scrotum, pituitary gland, pleura,prostate gland, rectum, renal pelvis & ureter, retroperitoneum &peritoneum, salivary gland, skin, small intestine, stomach, testis,thymus, thyroid gland, tongue, unknown, unspecified digestive organs,urinary bladder, uterus, nos, vagina & labia, and vulva, nos.

For example, in 168 individuals with advanced breast cancer (FIG. 30C),microarray analysis of 63 genes showed that the genes analyzed wereeither overexpressed or underexpressed a total of 1863 times and that5.05% of that total change in expression was attributable to SSTR3change in expression followed by 4.83% of the change in expression beingattributable to NKFBIA change in expression and 4.62% of the change inexpression being attributable to VDR. In addition, 4.35% of the changein expression was attributable to MGMT change in expression, 4.19% ofthe change in expression was attributable to ADA change in expression,and 3.97% of the change in expression was attributable to CES2 change inexpression.

FIG. 31 depicts a table showing biomarkers as targets in order offrequency in all tissues that were tested.

Example 4 A Study Using Molecular Profiling of Patients' Tumors to FindTargets and Select Treatments for Refractory Cancers

The primary objective was to compare progression free survival (PFS)using a treatment regimen selected by molecular profiling with the PFSfor the most recent regimen the patient progressed on (e.g. patients aretheir own control) (FIG. 32). The molecular profiling approach wasdeemed of clinical benefit for the individual patient who had a PFSratio (PFS on molecular profiling selected therapy/PFS on prior therapy)of ≧1.3.

The study was also performed to determine the frequency with whichmolecular profiling by IHC, FISH and microarray yielded a target againstwhich there is a commercially available therapeutic agent and todetermine response rate (RECIST) and percent of patients withoutprogression or death at 4 months.

The study was conducted in 9 centers throughout the United States. Anoverview of the method is depicted in FIG. 33. As can be seen in FIG.33, the patient was screened and consented for the study. Patienteligibility was verified by one of two physician monitors. The samephysicians confirmed whether the patients had progressed on their priortherapy and how long that PFS (TTP) was. A tumor biopsy was thenperformed, as discussed below. The tumor was assayed using IHC, FISH (onparaffin-embedded material) and microarray (on fresh frozen tissue)analyses.

The results of the IHC/FISH and microarray were given to two studyphysicians who in general used the following algorithm in suggestingtherapy to the physician caring for the patient: 1) IHC/FISH andmicroarray indicated same target was first priority; 2) IHC positiveresult alone next priority; and 3) microarray positive result alone thelast priority.

The patient's physician was informed of the suggested treatment and thepatient was treated with the suggested agent(s) (package insertrecommendations). The patient's disease status was assessed every 8weeks and adverse effects were assessed by the NCI CTCAE version 3.0.

To be eligible for the study, the patient was required to: 1) provideinformed consent and HIPAA authorization; 2) have any histologic type ofmetastatic cancer; 3) have progressed by RECIST criteria on at least 2prior regimens for advanced disease; 4) be able to undergo a biopsy orsurgical procedure to obtain tumor samples; 5) be ≧18 years, have a lifeexpectancy >3 months, and an Eastern Cooperative Oncology Group (ECOG)Performance Status or 0-1; 6) have measurable or evaluable disease; 7)be refractory to last line of therapy (documented disease progressionunder last treatment; received ≧6 weeks of last treatment; discontinuedlast treatment for progression); 8) have adequate organ and bone marrowfunction; 9) have adequate methods of birth control; and 10) if CNSmetastases then adequately controlled. The ECOG performance scale isdescribed in Oken, M. M., Creech, R. H., Tormey, D. C., Horton, J.,Davis, T. E., McFadden, E. T., Carbone, P. P.: Toxicity And ResponseCriteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol5:649-655, 1982, which is incorporated by reference in its entirety.Before molecular profiling was performed, the principal investigator atthe site caring for the patient must designate what they would treat thepatient with if no molecular profiling results were available.

Methods

All biopsies were performed at local investigators' sites. For needlebiopsies, 2-3 18 gauge needle core biopsies were performed. For DNAmicroarray (MA) analysis, tissue was immediately frozen and shipped ondry ice via FedEx to a central CLIA certified laboratory, Caris MPI inPhoenix, Ariz. For IHC, paraffin blocks were shipped on cold packs. IHCwas considered positive for target if 2+ in ≧30% of cells. The MA wasconsidered positive for a target if the difference in expression for agene between tumor and control organ tissue was at a significance levelof p≦0.001.

Ascertainment of the Time to Progression to Document theProgression-Free Survival Ratio

Time to progression under the last line of treatment was documented byimaging in 58 patients (88%). Among these 58 patients, documentation byimaging alone occurred in 49 patients (74%), and documentation byimaging with tumor markers occurred in nine patients (14%; ovariancancer, n 3; colorectal, n 1; pancreas, n 1; prostate, n 3; breast, n1). Patients with clinical proof of progression were accepted when theinvestigator reported the assessment of palpable and measurable lesions(i.e., inflammatory breast cancer, skin/subcutaneous nodules, or lymphnodes), which occurred in six patients (9%). One patient (2%) withprostate cancer was included with progression by tumor marker. In onepatient (2%) with breast cancer, the progression was documented byincrease of tumor marker and worsening of bone pain. The time toprogression achieved with a treatment based on molecular profiling wasdocumented by imaging in 44 patients (67%) and by clinical eventsdetected between two scheduled tumor assessments in 20 patients. Theseclinical events were reported as serious adverse events related todisease progression (e.g., death, bleeding, bowel obstruction,hospitalization), and the dates of reporting were censored asprogression of disease. The remaining two patients were censored at thedate of last follow-up.

IHC/FISH

For IHC studies, the formalin fixed, paraffin embedded tumor samples hadslices from these blocks submitted for IHC testing for the followingproteins: EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP, RRM1,TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase,Her2/neu and TOPO2A. IHCs for all proteins were not carried out on allpatients' tumors.

Formalin-fixed paraffin-embedded patient tissue blocks were sectioned (4μm thick) and mounted onto glass slides. After deparaffination andrehydration through a series of graded alcohols, pretreatment wasperformed as required to expose the targeted antigen.

Human epidermal growth factor receptor 2 (HER2) and epidermal growthfactor receptor (EGFR) were stained as specified by the vendor (DAKO,Denmark). All other antibodies were purchased from commercial sourcesand visualized with a DAB biotin-free polymer detection kit. Appropriatepositive control tissue was used for each antibody. Negative controlslides were stained by replacing the primary antibody with anappropriately matched isotype negative control reagent. All slides werecounterstained with hematoxylin as the final step and cover slipped.Tissue microarray sections were analyzed by FISH for EGFR and HER-2/neucopy number per the manufacturer's instructions. FISH for HER-2/neu (wasdone with the PathVysion HER2DNA Probe Kit (Abbott Molecular, AbbottPark, Ill.). FISH for EGFR was done with the LSI EGFR/CEP 7 Probe(Abbott Molecular).

All slides were evaluated semi-quantitatively by a first pathologist,who confirmed the original diagnosis as well as read each of theimmunohistochemical stains using a light microscope. Some lineageimmunohistochemical stains were performed to confirm the originaldiagnosis, as necessary. Staining intensity and extent of staining weredetermined; both positive, tumor-specific staining of tumor cells andhighly positive (≧2+), pervasive (≧30%) tumor specific staining resultswere recorded. IHC was considered positive for target if staining was≧2+ in ≧30% of cells. Rather than look for a positive signal withoutqualification, this approach raises the stringency of the cut point suchthat it would be a significant or more demonstrative positive. A higherpositive is more likely to be associated with a therapy that wouldaffect the time to progression. The cut point used (i.e., staining was≧2+ in ≧30% of cells) is similar to some cut points used in breastcancer for HER2/neu. When INC cut points were compared with evidencefrom the tissue of origin of the cancer, the cut points were equal to orhigher (more stringent) than the evidence cut points. A standard 10%quality control was performed by a second pathologist.

Microarray

Tumor samples obtained for microarray were snap frozen within 30 minutesof resection and transmitted to Caris-MPI on dry ice. The frozen tumorfragments were placed on a 0.5 mL aliquot of frozen 0.5M guanidineisothiocyanate solution in a glass tube, and simultaneously thawed andhomogenized with a Covaris S2 focused acoustic wave homogenizer(Covaris, Woburn, Mass.). A 0.5 mL aliquot of TriZol was added, mixedand the solution was heated to 65° C. for 5 minutes then cooled on iceand phase separated by the addition of chloroform followed bycentrifugation. An equal volume of 70% ethanol was added to the aqueousphase and the mixture was chromatographed on a Qiagen RNeasy column(Qiagen, Germantown, Md.). RNA was specifically bound and then eluted.The RNA was tested for integrity by assessing the ratio of 28S to 18Sribosomal RNA on an Agilent BioAnalyzer (Agilent, Santa Clara, Calif.).Two to five micrograms of tumor RNA and two to five micrograms of RNAfrom a sample of a normal tissue representative of the tumor's tissue oforigin were separately converted to cDNA and then labeled during T7polymerase amplification with contrasting fluor tagged (Cy3, Cy5)cytidine triphosphate. The labeled tumor and its tissue of originreference were hybridized to an Agilent HlAv2 60-mer olio array chipwith 17,085 unique probes.

The arrays contain probes for 50 genes for which there is a possibletherapeutic agent that would potentially interact with that gene (witheither high expression or low expression). Those 50 genes included: ADA,AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3,ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1,MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN,PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B,TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70.

The chips were hybridized from 16 to 18 hours at 60° C. and then washedto remove non-stringently hybridized probe and scanned on an AgilentMicroarray Scanner. Fluorescent intensity data were extracted,normalized, and analyzed using Agilent Feature Extraction Software. Geneexpression was judged to be different from its reference based on anestimate of the significance of the extent of change, which wasestimated using an error model that takes into account the levels ofsignal to noise for each channel, and uses a large number of positiveand negative controls replicated on the chip to condition the estimate.Expression changes at the level of p≦0.001 were considered assignificantly different.

Statistical Considerations

The protocol called for a planned 92 patients to be enrolled of which anestimated 64 patients would be treated with therapy assigned bymolecular profiling. The other 28 patients were projected to not havemolecular profiling results available because of (a) inability to biopsythe patient; (b) no target identified by the molecular profiling; or (c)deteriorating performance status. Sixty four patients were required toreceive molecular profiling treatment in order to reject the nullhypothesis (Ho) that: ≦15% of patients would have a PFS ratio of ≧1.3(e.g. a non-promising outcome).

Treatment Selection

Treatment for the patients based on molecular profiling results wasselected using the following algorithm: 1) IHC/FISH and microarrayindicates same target; 2) IHC positive result alone; 3) microarraypositive result alone. The patient's physician was informed of suggestedtreatment and the patient was treated based on package insertrecommendations. Disease status was assessed every 8 weeks. Adverseeffects were assessed by NCI CTCAE version 3.0.

The targets and associated drugs are listed in Table 17.

TABLE 17 Pairings of Targets and Drugs Potential Target Agents Suggestedas Interacting With the Target IHC EGFR Cetuximab, erlotinib, gefitinibSPARC Nanoparticle albumin-bound paclitaxel c-KIT Imatinib, sunitinib,sorafenib ER Tamoxifen, aromatase inhibitors, toremifene, progestationalagent PR Progestational agents, tamoxifen, aromatase inhibitor,goserelin Androgen receptor Flutamide, abarelix, bicalutamide,leuprolide, goserelin PGP Avoid natural products, doxorubicin,etoposide, docetaxel, vinorelbine HER2/NEU Trastuzumab PDGFR Sunitinib,imatinib, sorafenib CD52 Alemtuzumab CD25 Denileukin diftitox HSP90Geldanamycin, CNF2024 TOP2A Doxorubicin, epirubicin, etoposideMicroarray ADA Pentostatin, cytarabine AR Flutamide, abarelix,bicalutamide, leuprolide, goserelin ASNA Asparaginase BCL2 Oblimersensodium† BRCA2 Mitomycin CD33 Gemtuzumab ozogamicin CDW52 AlemtuzumabCES-2 Irinotecan DCK Gemcitabine DNMT1 Azacitidine, decitabine EGFRCetuximab, erlotinib, gefitinib ERBB2 Trastuzumab ERCC1 Cisplatin,carboplatin, oxaliplatin ESR1 Tamoxifen, aromatase inhibitors,toremifene, progestational agent FOLR2 Methotrexate, pemetrexed GARTPemetrexed GSTP1 Platinum HDAC1 Vorinostat HIF1α Bevacizumab, sunitinib,sorafenib HSPCA Geldanamycin, CNF2024 IL2RA Aldesleukin KIT Imatinib,sunitinib, sorafenib MLH-1 Gemcitabine, oxaliplatin MSH1 GemcitabineMSH2 Gemcitabine, oxaliplatin NFKB2 Bortezomib NFKB1 Bortezomib OGFROpioid growth factor PDGFC Sunitinib, imatinib, sorafenib PDGFRASunitinib, imatinib, sorafenib PDGFRB Sunitinib, imatinib, sorafenib PGRProgestational agents, tamoxifen, aromatase inhibitors, goserelin POLACytarabine PTEN Rapamycin (if low) PTGS2 Celecoxib RAF1 Sorafenib RARABexarotene, all-trans-retinoic acid RXRB Bexarotene SPARC Nanoparticlealbumin-bound paclitaxel SSTR1 Octreotide TK1 Capecitabine TNFInfliximab TOP1 Irinotecan, topotecan TOP2A Doxorubicin, etoposide,mitoxantrone TOP2B Doxorubicin, etoposide, mitoxantrone TXNRD1 Px12 TYMSFluorouracil, capecitabine VDR Calcitriol VEGF Bevacizumab, sunitinib,sorafenib VHL Bevacizumab, sunitinib, sorafenib ZAP70 Geldanamycin,CNF2024

Results

The distribution of the patients is diagrammed in FIG. 34 and thecharacteristics of the patients shown in Tables 18 and 19. As can beseen in FIG. 34, 106 patients were consented and evaluated. There were20 patients who did not proceed with molecular profiling for the reasonsoutlined in FIG. 34 (mainly worsening condition or withdrawing theirconsent or they did not want any additional therapy). There were 18patients who were not treated following molecular profiling (mainly dueto worsening condition or withdrawing consent because they did not wantadditional therapy). There were 68 patients treated, with 66 of themtreated according to molecular profiling results and 2 not treatedaccording to molecular profiling results. One of the two was treatedwith another agent because the clinician caring for the patient felt asense of urgency to treat and the other was treated with another agentbecause the insurance company would not cover the molecular profilingsuggested treatment.

The median time for molecular profiling results being made accessible toa clinician was 16 days from biopsy (range 8 to 30 days) and a median of8 days (range 0 to 23 days) from receipt of the tissue sample foranalysis. Some modest delays were caused by the local teams not sendingthe patients' blocks immediately (due to their need for a pathologyworkup of the specimen). Patient tumors were sent from 9 sitesthroughout the United States including: Greenville, S.C.; Tyler, Tex.;Beverly Hills, Calif.; Huntsville, Ala.; Indianapolis, Ind.; SanAntonio, Tex.; Scottsdale, Ariz. and Los Angeles, Calif.

Table 18 details the characteristics of the 66 patients who hadmolecular profiling performed on their tumors and who had treatmentaccording to the molecular profiling results. As seen in Table 17, ofthe 66 patients the majority were female, with a median age of 60 (range27-75). The number of prior treatment regimens was 2-4 in 53% ofpatients and 5-13 in 38% of patients. There were 6 patients (9%), whohad only 1 prior therapy because no approved active 2^(nd) line therapywas available. Twenty patients had progressed on prior phase Itherapies. The majority of patients had an ECOG performance status of 1.

TABLE 18 Patient Characteristics (n = 66) Characteristic n % GenderFemale 43 65 Male 23 35 Age Median (range) 60 (27-75) Number of PriorTreatments 2-4* 35 53 5-13 25 38 ECOG 0 18 27 1 48 73 *Note: 6 patients(9%) had 1 prior

As seen in Table 19, tumor types in the 66 patients included breastcancer 18 (27%), colorectal 11 (17%), ovarian 5 (8%), and 32 patients(48%) were in the miscellaneous categories. Many patients had the morerare types of cancers.

TABLE 19 Patient Tumor Types (n = 66) Tumor Type n % Breast 18 27Colorectal 11 17 Ovarian 5 8 Miscellaneous 32 48 Prostate 4 6 Lung 3 5Melanoma 2 3 Small cell (esopha/retroperit) 2 3 Cholangiocarcinoma 2 3Mesothelioma 2 3 H&N (SCC) 2 3 Pancreas 2 3 Pancreas neuroendocrine 11.5 Unknown (SCC) 1 1.5 Gastric 1 1.5 Peritoneal pseudomyxoma 1 1.5 AnalCanal (SCC) 1 1.5 Vagina (SCC) 1 1.5 Cervis 1 1.5 Renal 1 1.5 Eccrineseat adenocarinoma 1 1.5 Salivary gland adenocarinoma 1 1.5 Soft tissuesarcoma (uterine) 1 1.5 GIST (Gastric) 1 1.5 Thyroid-Anaplastic 1 1.5

Primary Endpoint: PFS Ratio ≧1.3

As far as the primary endpoint for the study is concerned (PFS ratio of≧1.3), in the 66 patients treated according to molecular profilingresults, the number of patients with PFS ratio greater or equal to 1.3was 18 out of the 66 or 27%, 95% CI 17-38% one-sided, one-sample nonparametric test p=0.007. The null hypothesis was that ≦15% of thispatient population would have a PFS ratio of ≧1.3. Therefore, the nullhypothesis is rejected and our conclusion is that this molecularprofiling approach is beneficial. FIG. 35 details the comparison of PFSon molecular profiling therapy (the bar) versus PFS (TTP) on thepatient's last prior therapy (the boxes) for the 18 patients. The medianPFS ratio is 2.9 (range 1.3-8.15).

If the primary endpoint is examined, as shown in Table 20, a PFS ratio≧1.3 was achieved in 8/18 (44%) of patients with breast cancer, 4/11(36%) patients with colorectal cancer, 1/5 (20%) of patients withovarian cancer and 5/32 (16%) patients in the miscellaneous tumor types(note that miscellaneous tumor types with PFS ratio ≧1.3 included: lung1/3, cholangiocarcinoma 1/3, mesothelioma 1/2, eccrine sweat gland tumor1/1, and GIST (gastric) 1/1).

TABLE 20 Primary Endpoint - PFS Ratio ≧1.3 By Tumor Type Tumor TypeTotal Treated Number with PFS Ratio ≧1.3 % Breast 18 8 44 Colorectal 114 36 Ovarian 5 1 20 Miscellaneous* 32 5 16 Total 66 18 27 *lung ⅓,cholangiocarcinoma ½, mesothelioma ½, eccrine sweat 1/1, GIST (gastric)1/1

The treatment that the 18 patients with the PFS ≧1.3 received based onprofiling is detailed in Table 21. As can be seen in that table forbreast cancer patients, the treatment ranged from diethylstibesterol tonab paclitaxel+gemcitabine to doxorubicin. Treatments for patients withother tumor types are also detailed in Table 21. The table further showsa comparison of the drugs that the responding patients received versusthe drugs that would have been suggested without molecular profiling andindicates which targets were used to suggest the therapies. Overall, 14were treated with combinations and 4 were treated with single agents.

TABLE 21 Targets Noted in Patients' Tumors, Treatment Suggested on theBasis of These Results, and Treatment Investigator Would Use if NoTarget Was Identified (in patients with PFS ratio ≧1.3) Treatment theTreatment Suggested Investigator Would Targets Used to on Basis ofPatient's Have Used if No Location of Primary Suggest Treatment TumorMolecular Results From Tumor and Method Used Profiling MolecularProfiling Breast ESR1: I; ESR1: M DES 5 mg TID InvestigationalCholangiocarcinoma EGFR: I; TOP1: M CPT-11 350 mg/m² Investigationalevery 3 weeks; cetuximab 400 mg/m² day 1, 250 mg/m² every week BreastSPARC: I; SPARC, NAB paclitaxel 260 mg/m² Docetaxel, trastuzumab ERBB2:M every 3 weeks; trastuzumab 6 mg/kg every 3 weeks Eccrine sweat glandc-KIT: I; c-KIT:M Sunitinib 50 mg/d, 4 Best supportive care (rightforearm) weeks on/2 weeks off Ovary HER2/NEU, ER: I; Lapatinib 1,250 mgPO Bevacizumab HER2/NEU: M days 1-21; tamoxifen 20 mg PO Colon/rectumPDGFR, c-KIT: I I; CPT-11 70 mg/m² Cetuximab PDGFR, TOP1: M weekly for 4weeks on/2 weeks off; sorafenib 400 mg BID Breast SPARC: I; DCK: M NABpaclitaxel 90 mg/m² Mitomycin every 3 weeks; gemcitabine 750 mg/m² days1, 8, 15, every 3 weeks Breast ER: I; ER, TYMS: M Letrozole 2.5 mgdaily; Capecitabine capecitabine 1,250 mg/m² BID, 2 weeks on/1 week offMalignant mesothelioma MLH1, MLH2: I; Gemcitabine 1,000 mg/m2Gemcitabine RRM2B, RRM1, RRM2, days 1 and 8, TOP2B: M every 3 weeks;etoposide 50 mg/m² 3 days every 3 weeks Breast MSH2 Oxaliplatin 85 mg/m²Investigational every 2 weeks; fluorouracil (5FU) 1,200 mg/m² days 1 and2, every 2 weeks; trastuzumab 4 mg/kg day 1, 2 mg/kg every weekNon-small-cell lung EGFR: I; EGFR Cetuximab 400 mg/m² Vinorelbine cancerday 1, 250 mg/m² every week; CPT-11 125 mg/m² weekly for 4 weeks on/2weeks off Colon/rectum MGMT Temozolomide 150 mg/m² Capecitabine for 5days every 4 weeks; bevacizumab 5 mg/kg every 2 weeks Colon/rectumPDGFR, c-KIT: I; Mitomycin 10 mg once Capecitabine PDGFR: KDR, HIF1A,every 4-6 weeks; BRCA2: M sunitinib 37.5 mg/d, 4 weeks on/2 weeks offBreast DCK, DHFR: M Gemcitabine 1,000 mg/m² Best supportive care days 1and 8 every 3 weeks; pemetrexed 500 mg/m² days 1 and 8, every 3 weeksBreast TOP2A: I; TOP2A: M Doxorubicin 50 mg/m² Vinorelbine every 3 weeksColon/rectum MGMT, VEGFA, Temozolomide 150 mg/m² Panitumumab HIF1A: Mfor 5 days every 4 weeks; sorafenib 400 mg BID Breast ESR1, PR: I; ESR1,PR: M Exemestane 25 mg Doxorubicin liposomal every day GIST (stomach)EGFR: I; EGFR, Gemcitabine 1,000 mg/m² None RRM2: M days 1, 8, and 15every 4 weeks; cetuximab 400 mg/m² day 1, 250 mg/m² every week*Abbreviations used in Table 21: I, immunohistochemistry; M, microarray;DES, diethylstilbestrol; CPT-11, irinotecan; TID, three times a day;NAB, nanoparticle albumin bound; PO, orally; BID, twice a day; GIST, GIstromal tumor.

Secondary Endpoints

The results for the secondary endpoint for this study are as follows.The frequency with which molecular profiling of a patients' tumoryielded a target in the 86 patients where molecular profiling wasattempted was 84/86 (98%). Broken down by methodology, 83/86 (97%)yielded a target by IHC/FISH and 81/86 (94%) yielding a target bymicroarray. RNA was tested for integrity by assessing the ratio of 28Sto 18S ribosomal RNA on an Agilent BioAnalyzer. 83/86 (97%) specimenshad ratios of 1 or greater and gave high intra-chip reproducibilityratios. This demonstrates that very good collection and shipment ofpatients' specimens throughout the United States and excellent technicalresults can be obtained.

By RECIST criteria in 66 patients, there was 1 complete response and 5partial responses for an overall response rate of 10% (one CR in apatient with breast cancer and PRs in breast, ovarian, colorectal andNSCL cancer patients). Patients without progression at 4 months included14 out of 66 or 21%.

In an exploratory analysis, a waterfall plot for all patients formaximum % change of the summed diameters of target lesions with respectto baseline diameters was generated. The patients who had progressionand the patients who had some shrinkage of their tumor sometime duringtheir course along with those partial responses by RECIST criteria isdemonstrated in FIG. 36. There is some shrinkage of patient's tumors inover 47% of the patients (where 2 or more evaluations were completed).

Other Analyses—Safety

As far as safety analyses there were no treatment related deaths. Therewere nine treatment related serious adverse events including anemia (2patients), neutropenia (2 patients), dehydration (1 patient),pancreatitis (1 patient), nausea (1 patient), vomiting (1 patient), andfebrile neutropenia (1 patient). Only one patient (1.5%) wasdiscontinued due to a treatment related adverse event of grade 2fatigue.

Other Analyses—Relationship Between What the Clinician Caring for thePatient would have Selected Versus What the Molecular Profiling Selected

The relationship between what the clinician selected to treat thepatient before knowing what molecular profiling results suggested fortreatment was also examined. As detailed in FIG. 37, there is no patternbetween the two. More specifically, no matches for the 18 patients withPFS ratio ≧1.3 were noted.

The overall survival for the 18 patients with a PFS ratio of ≧1.3 versusall 66 patients is shown in FIG. 38. This exploratory analysis was doneto help determine if the PFS ratio had some clinical relevance. Theoverall survival for the 18 patients with the PFS ratio of ≧1.3 is 9.7months versus 5 months for the whole population— log rank 0.026. Thisexploratory analysis indicates that the PFS ratio is correlated with theclinical parameter of survival.

Conclusions

This prospective multi-center pilot study demonstrates: (a) thefeasibility of measuring molecular targets in patients' tumors from 9different centers across the US with good quality and sufficient tumorcollection—and treat patients based on those results; (b) this molecularprofiling approach gave a longer PFS for patients on a molecularprofiling suggested regimen than on the regimen they had just progressedon for 27% of the patients (confidence interval 17-38%) p=0.007; and (c)this is a promising result demonstrating use and benefits of molecularprofiling.

The results also demonstrate that patients with refractory cancer cancommonly have simple targets (such as ER) for which therapies areavailable and can be beneficial to them. Molecular profiling forpatients who have exhausted other therapies and who are perhapscandidates for phase I or II trials could have this molecular profilingperformed.

Example 5 Molecular Profiling System

Molecular profiling is performed to determine a treatment for a disease,typically a cancer. Using a molecular profiling approach, molecularcharacteristics of the disease itself are assessed to determine acandidate treatment. Thus, this approach provides the ability to selecttreatments without regard to the anatomical origin of the diseasedtissue, or other “one-size-fits-all” approaches that do not take intoaccount personalized characteristics of a particular patient'saffliction. The profiling comprises determining gene and gene productexpression levels, gene copy number and mutation analysis. Treatmentsare identified that are indicated to be effective against diseased cellsthat overexpress certain genes or gene products, underexpress certaingenes or gene products, carry certain chromosomal aberrations ormutations in certain genes, or any other measurable cellular alterationsas compared to non-diseased cells. Because molecular profiling is notlimited to choosing amongst therapeutics intended to treat specificdiseases, the system has the power to take advantage of any usefultechnique to measure any biological characteristic that can be linked toa therapeutic efficacy. The end result allows caregivers to expand therange of therapies available to treat patients, thereby providing thepotential for longer life span and/or quality of life than traditional“one-size-fits-all” approaches to selecting treatment regimens.

A molecular profiling system has several individual components tomeasure expression levels, chromosomal aberrations and mutations. Thecomponents are shown in FIG. 39. These include immunohistochemistryassays (IHC) on formalin fixed paraffin embedded (FFPE) cancer tissue.To perform IHC on a sample, a paraffin embedded block with a largesection of tumor (at least 20% viable neoplasm) from the procedure whichis preferred. For any tumor, IHC is run for 18 target genes comprisingdruggable or drug resistant targets. IHC can be performed on additionalgenes depending on disease characteristics, e.g., tumor origin andprogression. In addition to IHC, gene expression arrays, such as theAgilent 44K chip (Agilent Technologies, Inc., Santa Clara, Calif.). Thissystem is capable of determining the relative expression level ofroughly 44,000 different sequences through RT-PCR from RNA extractedfrom fresh frozen tissue. The expression of 80 druggable or drugresistant targets is examined in further detail. Because of thepracticalities involved in obtaining fresh frozen tissue, only a portionof samples with sufficient quantity and quality of mRNA are analyzedusing microarray analysis. The system also assesses gene copy numberand/or other chromosomal abnormalities for a number of genes using FISH(fluorescence in situ hybridization). Finally, mutation analysis is doneby DNA sequencing for a several specific mutations. All of this data isstored for each patient case. Microarray results IHC, FISH and DNAsequencing analysis for a number of genes that have been shown to impacttherapeutic options are used to generate a final patient report. Thereport can include a prioritized list of druggable targets and theirassociated therapies. The report is explained by a practicingoncologist. Once the data are reported, the final decisions rest withthe treating physician. Based on this approach, the treating physicianhas information on therapies that might not otherwise have beenconsidered based on the lineage of the disease.

Example 6 Illumina Expression Analysis

The Illumina Whole Genome DASL assay (Illumina Inc., San Diego, Calif.)offers a method to simultaneously profile over 24,000 transcripts fromminimal RNA input, from both fresh frozen (FF) and formalin-fixedparaffin embedded (FFPE) tissue sources, in a high throughput fashion.The analysis makes use of the Whole-Genome DASL Assay with UDG(Illumina, cat#DA-903-1024/DA-903-1096), the Illumina HybridizationOven, and the Illumina iScan System.

A small piece (0.25 gm-0.5 gm) of tumor or 4-5 cores flash-frozen within30 minutes of extraction from the patient is preferred to preserve theRNA. This tissue is preferably preservative-free (e.g., no exposure toalcohol) and remains frozen (e.g., either in a −80° freezer or on dryice once frozen). If fresh tissue is not available, one paraffin block(40% Tumor) or 45 unstained slides can be used. The sample can betreated to preserve the RNA, e.g., using RNAlater® RNA stabilizationsolution according to the manufacturer's instructions (AppliedBiosystems/Ambion, Austin, Tex.). The RNA preservative stabilizationsolution is an aqueous tissue storage reagent that rapidly permeatesmost tissues to stabilize and protect RNA in fresh specimens. Samples inRNA Preservative solution can be stored for periods of time that mayotherwise render RNA unusable for molecular profile assays.

The Whole Genome DASL assay is performed following the manufacturer'sinstructions. Total RNA isolated from either FF or FFPE sources isconverted to cDNA using biotinylated oligo(dT) and random nonamerprimers. The use of both oligo(dT) and random nonamer primers helpsensure cDNA synthesis of degraded RNA fragments, such as those obtainedfrom FFPE tissue. The biotinylated cDNA is then annealed to the DASLAssay Pool (DAP) probe groups. Probe groups contain oligonucleotidesspecifically designed to interrogate each target sequence in thetranscripts. The probes span around 50 bases, allowing for the profilingof partially degraded RNA.

The assay probe set consists of an upstream oligonucleotide containing agene specific sequence and a universal PCR primer sequence (P1) at the5′ end, and a downstream oligonucleotide containing a gene specificsequence and a universal PCR primer sequence (P2) at the 3′ end. Theupstream oligonucleotide hybridizes to the targeted cDNA site, and thenextends and ligates to its corresponding downstream oligonucleotide tocreate a PCR template that can be amplified with universal PCR primersaccording to the manufacturer's instructions.

The resulting PCR products are hybridized to the HumanRef-8 ExpressionBeadChip to determine the presence or absence of specific genes. TheHumanRef-8 BeadChip features up-to-date content covering >24,000annotated transcripts derived from the National Center for BiotechnologyInformation Reference Sequence (RefSeq) database (Build 36.2, Release22). For details see Tables 22 and 23.

TABLE 22 HumanRef-8 Expression Array Characteristic Number Transcripts24,526 Genes 18,401 Probe Beads ~1,000,000 Probe Beads/Transcript ~41Control Probes ~850 Probes for 50-base site on transcript Two 25-mers

TABLE 23 RefSeq* Content of the HumanRef-8 BeadChip Probes DescriptionNumber NM Coding transcripts, well established annotations 23,811 XMCoding transcripts, provisional annotations 426 NR Non-codingtranscripts, well established annotations 263 XR Non-coding transcripts,provisional annotations 26 Total 24,526 *Build 36.2, Release 22

After hybridization, HumanRef-8 Expression BeadChips are scanned usingthe iScan system. This system incorporates high-performance lasers,optics, and detection systems for rapid, quantitative scanning. Thesystem offers a high signal-to-noise ratio, high sensitivity, low limitof detection, and broad dynamic range, leading to exceptional dataquality.

Whole genome gene expression analysis using DASL chemistry microarraysallows for an estimate of whether a particular gene is producing more orless mRNA in the tumor than in the cell type from which the tumor wasderived. Based on the activity, greater or lesser, of a given gene, mayincrease the likelihood that a tumor will respond to a particulartherapeutic depending on the type of cancer being treated. Thedifferential gene expression of a subject's tumor when compared tonormal tissue can provide a useful diagnostic tool for helping anoncologist determine the appropriate treatment route.

The DASL chemistry addresses the limitation of working with degradedFFPE RNA by deviating from the traditional direct hybridizationmicroarray methodologies. However, there is much variability in fixationmethods of FFPE tissue, which can lead to higher levels of RNAdegradation. The DASL assay can be used for partially degraded RNAs, butnot for entirely degraded RNAs. To qualify RNA samples prior to DASLassay analysis, RNA quality is checked using a real-time qPCR methodwhere the highly expressed ribosomal protein gene, RPL13a, is amplifiedusing SYBR green chemistry. If a sample has a cycle threshold value ≦29,then the sample is considered to be intact enough to proceed with theDASL chemistry. See Biotinylated cDNA Pre-Qualification, Illumina, Inc.;Abramovitz, M., et al., Optimization of RNA extraction from FFPE tissuesfor expression profiling in the DASL assay. Biotechniques, 2008. 44(3):p. 417-23. Any sample that has an A260/A280 ratio <1.5, or a RPL13a Ctvalue >30 is considered too degraded or too heavily modified to beprocessed using the Whole Genome DASL gene expression chemistry. SeeAbramovitz.

Prior to hybridization on the HumanRef-8 Expression BeadChip, the sampleis precipitated. The sample precipitate will be in the form of a bluepellet. If the blue pellet is not visible for that sample, the samplemust be re-processed prior to hybridization on the BeadChip.

Although the Whole Genome DASL assay examines the expression ofthousands of genes, expression of only the genes of interest need beanalyzed.

In order to standardize the reporting of patient data using the IlluminaWhole Genome DASL technology, the algorithm below is used. The data isobtained using the Genome Studios Software v2009.1 (Gene ExpressionModule version 1.1.1).

Step 1:

The detection p-values determined by the Genome Studios software must beless than 0.01. This value is determined by examining the variability ofthe signals generated by the duplicate copies of the same probe for aparticular gene in relation to the variability observed in the negativecontrol probes present on the array. If the detection p-value for eitherthe control or the patient sample is greater than 0.01 for a particulargene the expression for that gene is reported out as “Indeterminate.” Acut-off of 0.01 was selected as it indicates that there is less than aone percent chance that the data would be observed given that the nullhypothesis of no change in expression is true. The p-value can becorrected for multiple comparisons.

Step 2:

The p-value of the differential expression must be less than 0.001. Thisp-value is determined by using the following equation:1/(10̂(D/(10*SIGN(PS−CS)))). In this equation “D” represents thedifferential expression score that is generated by the Genome Studios.The “PS” and “CS” represents the relative fluorescence units (RFU)obtained on the array of a particular gene for the patient sample (PS)and control sample (CS) respectively. The “SIGN” function converts thesign of the value generated by subtracting the CS RFU from the PS RFUinto a numerical value. If PS minus CS is >0 a value of 1 will begenerated. If PS minus CS is <0 a value of −1 will be generated. If PSequals CS then a value of 0 will be generated. If the differentialexpression p-value is greater than 0.001 for any particular gene theexpression for that gene is reported out as “No Change.” A cut off of0.001 was chosen because genes passing this threshold can be validatedas differentially expressed by alternative methods approximately 95% ofthe time.

Step 3:

If the expression ratio is less than 0.66 for a particular gene, theexpression for that gene will be reported out as “Underexpressed.” Ifthe expression ratio is greater than 1.5, the expression for that genewill be reported out as “Overexpressed.” If the expression ratio isbetween 0.66 and 1.5 the expression for a particular gene will bereported out as “No Change.” The expression ratio is determined byobtained by dividing the RFUs for a gene from the patient sample by theRFUs for the same gene from the control sample (PS/CS). “No Change”indicates that there is no difference in expression for this genebetween tumor and control tissues at a significance level of p<=0.001. Asignificance level of p<=0.001 was chosen since genes passing thisthreshold can be validated as differentially expressed by alternativemethods approximately 95% of the time.

“Not Informative (NI)” indicates that the data obtained for either thepatient sample or the control sample were not of high enough quality toconfidently make a call on the expression level of that particular RNAtranscript.

Step 4:

In some where FFPE samples only are used, all genes that are identifiedas “Under expressed”, using the above algorithm, will be reported out as“Indeterminate.” This is due to the degraded nature of the RNA obtainedfrom FFPE samples and as such, it may not be possible to determinewhether or not the reduced RFUs for a gene in the patient samplerelative to the control sample is due to the reduced presence of thatparticular RNA or if the RNA is highly degraded and impeding thedetection of that particular RNA transcript. With improved technologies,some or all genes as “Underexpressed” with FFPE samples are reported.

FIG. 40 shows results obtained from microarray profiling of an FFPEsample. Total RNA was extracted from tumor tissue and was converted tocDNA. The cDNA sample was then subjected to a whole genome (24K)microarray analysis using Illumina cDNA-mediated annealing, selection,extension and ligation (DASL) process. The expression of a subset of 80genes was then compared to a tissue specific normal control and therelative expression ratios of these 80 target genes indicated in thefigure was determined as well as the statistical significance of thedifferential expression.

Example 7 Molecular Profiling System and Report

A system has several individual components including a gene expressionarray using the Illumina Whole Genome DASL Assay as described in Example6. In addition to this gene expression array, the system also performs asubset of immunohistochemistry assays on formalin fixed paraffinembedded (FFPE) cancer tissue. Gene copy number is determined for anumber of genes via FISH (fluorescence in situ hybridization) andmutation analysis is done by DNA sequencing for a several specificmutations. All of this data is stored for each patient case. Data isreported from the microarray, IHC, FISH and DNA sequencing analysis. Alllaboratory experiments are performed according to Standard OperatingProcedures (SOPs).

DNA for mutation analysis is extracted from formalin-fixedparaffin-embedded (FFPE) tissues after macrodissection of the fixedslides in an area that % tumor nuclei ≧10% as determined by apathologist. Extracted DNA is only used for mutation analysis if % tumornuclei ≧10%. DNA is extracted using the QJAamp DNA FFPE Tissue kitaccording to the manufacturer's instructions (QIAGEN Inc., Valencia,Calif.). DNA can also be extracted using the QuickExtract™ FFPE DNAExtraction Kit according to the manufacturer's instructions (EpicentreBiotechnologies, Madison, Wis.). The BRAF Mutector I BRAF Kit (TrimGen,cat#MH1001-04) is used to detect BRAF mutations (TrimGen Corporation,Sparks, Md.). The DxS KRAS Mutation Test Kit (DxS, #KR-03) is used todetect KRAS mutations (QIAGEN Inc., Valencia, Calif.). BRAF and KRASsequencing of amplified DNA is performed using Applied Biosystems'BigDye® Terminator V1.1 chemistry (Life Technologies Corporation,Carlsbad, Calif.).

IHC is performed according to standard protocols. IHC detection systemsvary by marker and include Dako's Autostainer Plus (Dako North America,Inc., Carpinteria, Calif.), Ventana Medical Systems Benchmark® XT(Ventana Medical Systems, Tucson, Ariz.), and the Leica/VisionBiosystems Bond System (Leica Microsystems Inc., Bannockburn, Ill.). Allsystems are operated according to the manufacturers' instructions.American Society of Clinical Oncology (ASCO) and College of AmericanPathologist (CAP) standards are followed for ER, PR, and HER2 testing.ER, PR and HER2 as well as Ki-67, p53, and E-cad IHCs analyzed by theACIS® (Automated Cellular Imaging System). The ACIS system comprises amicroscope that scans the slides and constructs an image of the entiretissue section. Ten areas of tumor are analyzed for percentage positivecells and staining intensity within the selected fields.

FISH is performed on formalin-fixed paraffin-embedded (FFPE) tissue.FFPE tissue slides for FISH must be Hematoxylin and Eosin (H&E) stainedand given to a pathologist for evaluation. Pathologists will mark areasof tumor for FISH analysis. The pathologist report shows whether tumoris present and sufficient enough to perform a complete analysis. FISH isperformed using the Abbott Molecular VP2000 according to themanufacturer's instructions (Abbott Laboratories, Des Plaines, Iowa).

A report generated by the system in shown in FIGS. 41A-41J. FIG. 41Ashows that the patient had a primary tumor in the ovary. The patient isa 77 year-old female with a history of papillary serous carcinoma. Basedon the profiling, agents associated with clinical benefit on NCCNCompendiums included topotecan, irinotecan, paclitaxel, docetaxel,cisplatin, carboplatin, liposomal-doxorubicin, gemcitabine, bevacizumaband letrozole. Agents associated with clinical benefit off NCCNCompendium™ included doxorubicin, dasatinib, calcitriol,cholecalciferol, sorafenib, and sunitinib. Agents associated with a lackof clinical benefit based on the profiling include trastuzumab,temozolomide, methotrexate, pemetrexed, capecitabine. FIG. 41B showsthat the specimen consisted of a paraffin block. FIG. 41C presents atable of agents associated with benefits, and the biomarker analysismethod and results that indicated the beneficial agents. FIG. 41Dpresents a table of agents associated with lack of benefit, and thebiomarker analysis method and results that indicated these agents. FIG.41E presents the results of IHC analysis and FIG. 41F presents theresults of microarray analysis. FIG. 41G described microarraymethodology. FIG. 41H presents the results of FISH analysis. FIG. 41Ipresent a summary description of the differentially expressedbiomarkers. FIGS. 41J-41K present a summary description of literaturesupporting the candidate therapeutics linked to the differentiallyexpressed biomarkers with a rating for the level of evidence attached toeach publication. FIG. 41L presents a chart explaining the codes forlevel of evidence.

A second exemplary report is shown in FIGS. 42A-42J. FIG. 42A shows thatthe patient had a primary tumor in the ovary. The patient is a 74year-old female with a history of metastatic serous adenocarcinoma.Based on the profiling, agents associated with clinical benefit on NCCNCompendium™ included letrozole, anastrozole, tamoxifen, paclitaxel,docetaxel, cisplatin, carboplatin, liposomal-doxorubicin, and megestrolacetate. Agents associated with clinical benefit off NCCN Compendium™included aminoglutethimide, exemestane, fulvestrant, toremifene,doxorubicin, calcitriol, cholecalciferol, and medroxyprogesterone.Agents associated with a lack of clinical benefit based on the profilinginclude topotecan, irinotecan, trastuzumab, pemetrexed, andcapecitabine. FIG. 42B shows that the specimens consisted of a paraffinblock and tissue biopsy slide. FIG. 42C presents a table of agentsassociated with benefits, and the biomarker analysis method and resultsthat indicated the beneficial agents. FIG. 42D presents a table ofagents associated with lack of benefit, and the biomarker analysismethod and results that indicated these agents. FIG. 42E presents theresults of IHC analysis and FIG. 42F presents the results of microarrayanalysis. FIG. 42G described microarray methodology. FIG. 42H presentsthe results of FISH analysis. FIG. 42I present a summary description ofthe differentially expressed biomarkers. FIGS. 42J-42K present a summarydescription of literature supporting the candidate therapeutics linkedto the differentially expressed biomarkers with a rating for the levelof evidence attached to each publication. FIG. 42L presents a chartexplaining the codes for level of evidence.

Example 8 Workflow for Identifying a Therapeutic Agent

FIG. 43 illustrates a diagram that outlines a workflow for identifying atherapeutic agent by analyzing a sample from an individual (431). Thisexemplary workflow is presented with respect to breast cancer but one ofskill will appreciate that the workflow can be readily adapted for otherdisorders and cancers. The sample is cut into a number of slides (432)and stained with hematoxylin and eosin (H&E) (433). The stained slidesare read by a pathologist (434) to determine what panel of markers totest, e.g., whether to analyze the sample using a complete biomarkerpanel analysis or a tumor-specific biomarker panel analysis, e.g., forbreast cancer sample analysis (435). The pathologist also identifiessections (436) for DNA microarray analysis (437), FISH analysis, e.g.,to measure HER2 expression (438), or mutational analysis via sequencing(439). DNA microarray analysis can be performed on a whole genome scale,with focus on genes that are informative for therapeutic treatmentoptions, including at least ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5,BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DIEM, DNMT1, DNMT3A, DNMT3B,ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART,GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5,IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2,NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN,PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC,SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B,TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. IHC is run on selectedsections to analyze expression of biomarkers including AR, c-kit, CAV-1,CK 5/6, CK14, CK17, ECAD, ER, Her2/Neu, Ki67, MRP1, P53, PDGFR, PGP, PR,PTEN, SPARC, TLE3 and TS (4310). Each marker can be analyzed using asingle or multiple antibodies for IHC detection. For example, SPARC isdetected using an anti-SPARC monoclonal antibody (referred to herein asSPARC MC, SPARC Mono, SPARC m or the like), and an anti-SPARC polyclonalantibody (referred to herein as SPARC PC, SPARC Poly, SPARC p or thelike), Given the results of the previous analysis, the sample is furtheranalyzed with relevant marker panels (4311). The sample is classified asHER2+ (4312), Triple Negative (4316), or ER/PR+, HER2− (4318). Furtheranalysis depends on whether prior analysis determined that the sampleshould undergo “complete” biomarker panel analysis or a “tumor-specific”biomarker panel analysis. Tumor-specific analysis is performed for anycancer with a primary diagnosis, or first line, second line or thirdline therapy. Complete biomarker analysis is indicated for cancers thatare fourth line, metastatic or beyond. Complete is also performed if thetherapeutic history of the cancer is unknown (and thus becomes thedefault). In this manner, unnecessary testing can be avoided. HER2+(4312) samples are further analyzed by FISH for CMYC and TOP2A (4313),by IHC for p95 for tumor-specific analysis or for BCRP, ERCC1, MGMT,P95, RRM1, TOP2A and TOPO1 for complete analysis (4314), and bysequencing for mutation analysis of PIK3CA (4315). Triple negative(4316) samples are analyzed by IHC for p95 for tumor-specific analysisor for BCRP, ERCC1, MGMT, P95, RRM1, TOP2A and TOPO1 for completeanalysis (4317). ER/PR+, HER2− (4318) samples are further analyzed byFISH for CMYC (4319), by IHC for p95 for tumor-specific analysis or forBCRP, ERCC1, MGMT, P95, RRM1, TOP2A and TOPO1 for complete analysis(4320). The results of the analysis are used to identify a therapeuticfor the individual. The workflow can be generalized for the analysis ofother diseases and tumor types.

FIGS. 44A-B illustrate a biomarker centric view of the workflowdescribed above. In FIG. 44A, initial IHC and FISH results on theindicated biomarkers is used to characterize the cancer as HER2+, TripleNegative, or ER/PR+, HER2−. The characterization guides the additionalIHC, FISH and sequencing analysis that is performed. “DNA MA” indicatesthat a DNA microarray is performed on all samples that meet the qualitythreshold as described herein. DNA microarray analysis can be performedon a whole genome scale, with focus on genes that are informative fortherapeutic treatment options, including at least ABCC1, ABCG2, ADA, AR,ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR,DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1,FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1,IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1,MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB,PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B,RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTRS, TK1,TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70.IHC is run on selected sections to analyze expression of biomarkersincluding AR, c-kit, CAV-1, CK 5/6, CK14, CK17, ECAD, ER, Her2/Neu,Ki67, MRP1, P53, PDGFR, PGP, PR, PTEN, SPARC, TLE3 and TS. FIG. 44Boutline shows the criteria used to perform additional assays.Tumor-specific analysis is used in the case of cancer with a primarydiagnosis, or first line, second line or third line therapy. Completebiomarker analysis is indicated for cancers that are fourth line,metastatic or beyond.

Table 24 indicates prognostic markers in the breast cancer profiling.The markers used in the profiling can be used for theranostic (e.g., toguide selection of a candidate therapeutic) and prognostic purposes. “Y”in the “Prognostic?” column indicates that the marker can indicate aprognosis. Further details are described herein.

TABLE 24 Prognostic Breast Cancer Profiling Triple ER/PR+/ HER2+ NegHER2- Biomarker Method Prognostic? Profile Profile Profile AR IHC Y Y YCaveolin-1 IHC Y Y Y Y CK 14 IHC Y Y Y Y CK 17 IHC Y Y Y Y CK 5/6 IHC YY Y Y c-Kit IHC Y Y Y Y cMYC FISH Y Y Y Cyclin D1 IHC Y Y ECAD IHC Y Y YY EGFR IHC Y Y ER (ESR1) IHC Y Y Y HER2 IHC/FISH Y Y Y (ERBB2) Ki67 IHCY Y Y MRP1 IHC Y Y Y (ABCC1) P53 IHC Y Y Y Y P95 IHC Y Y Y PDGFR IHC Y YY Y PGP (ABCB1) IHC Y Y Y PI3K SEQ Y PR IHC Y Y Y PTEN IHC Y Y Y SPARCIHC Y Y Y TLE3 IHC Y Y Y TOP2A FISH Y TOP2A IHC Y Y Y TS (TYMS) IHC Y YY

Table 25 provides illustrative candidate treatments corresponding to themolecular profiling described in this Example. In the table, a positiveresult for the indicated biomarker using the indicated technique guidesselection of the corresponding therapeutic agent, or that of a relatedagent.

TABLE 25 Illustrative Drug-biomarker Associations Drug MethodBiomarker(s) 5-fluorouracil DNA Microarray TYMS IHC TS aminoglutethimideDNA Microarray ESR1, PR IHC ER, PR anastrozole DNA Microarray ESR1, PRIHC ER, PR capecitabine DNA Microarray TYMS IHC TS doxorubicin DNAMicroarray ABCB1, TOP2A FISH HER2, TOP2A IHC PGP, TOP2A epirubicin DNAMicroarray ABCB1, TOP2A FISH HER2, TOP2A IHC PGP, TOP2A exemestane DNAMicroarray ESR1, PR IHC ER, PR fulvestrant DNA Microarray ESR1, PR IHCER, Ki67, PR gonadorelin DNA Microarray PR goserelin DNA Microarray PRirinotecan IHC TOPO1 lapatinib FISH HER2 IHC HER2 letrozole DNAMicroarray ESR1, PR IHC ER, PR leuprolide DNA Microarray PRliposomal-doxorubicin DNA Microarray ABCB1, TOP2A FISH HER2, TOP2A IHCPGP, TOP2A medroxyprogesterone DNA Microarray ESR1, PR IHC ER, PRmegestrol acetate DNA Microarray ESR1, PR IHC ER, PR methotrexate DNAMicroarray ABCC1, DHFR IHC MRP1 nab-paclitaxel DNA Microarray SPARC IHCSPARC mono, SPARC poly pemetrexed DNA Microarray DHFR, GART, TYMS IHC TStamoxifen DNA Microarray ESR1, PR IHC ER, Ki67, PR taxanes IHC TLE3toremifene DNA Microarray ESR1, PR IHC ER, Ki67, PR trastuzumab FISHHER2 IHC HER2, P95, PTEN Mutation (sequence analysis) PIK3CA

An illustrative benefit of the molecular profiling approach isillustrated in FIG. 45. For every 100 HER2+ patients, only about 30(30%) will be Responders to treatment with trastuzumab. Molecularprofiling according to the Example identifies 50 (50%) out of the 70patients (70%) not likely to respond, e.g., because of PIK3CA mutations(25%), lack of PTEN (15%) or a p95 HER2 truncation (10%). HER2 spans thecell membrane and trastuzumab binds the external portion of the protein.However, most HER2 tests, including the FDA approved tests availablefrom Dako (Dako North America, Inc., Carpinteria, Calif.) and Ventana(Ventana Medical Systems, Inc., Tuscon, Ariz.), target the internaldomain of HER2. Profiling according to the invention uses two antibodiesfor HER2: one with affinity to the internal domain, another withaffinity to both the internal and external domains. If the latterantibody is negative but the tests targeting the internal domain arepositive (e.g., the FDA approved tests), then HER2 is “p95 truncated”and trastuzumab will not be effective. By identifying patients unlikelyto respond, efficacy of trastuzumab for a selected population can beincreased from 30% to 60%. Furthermore, the molecular profiling methodsof the invention can identify candidate treatments that are more likelyto be effective in the trastuzumab non-responders.

A patient profile report can be generated as in FIGS. 41 and 42.

Example 9 Biomarker and Drug-Centric Molecular Profiling

FIG. 46 illustrates a diagram showing a biomarker centric (FIG. 46A) andtherapeutic centric (FIG. 46B) approach to identifying a therapeuticagent. Mutational analysis is performed on the markers with symbols initalics. This typically comprises a sequencing approach (e.g., Sangersequencing or pyrosequencing) or an amplification approach (e.g., realtime PCR). ISH, e.g., FISH, is performed on the markers whose symbolsare underlined. The remaining markers are analyzed by IHC. DNAmicroarrays are performed on all samples with RNA of sufficient quality.In the biomarker-centric approach of FIG. 46A, the panel of markers thatare run on a sample to identify a candidate therapeutic can depend onthe origin of the tumor. Each circle surrounds the markers that areanalyzed for a cancer of the indicated origin. Markers analyzed forbreast cancers include FISH for cMYC and HER2, mutational analysis forPIK3CA, and IHC for P53, Ki67, p95, CK 14, CK 5/6, Cyclin D1, CAV-1,CK17, EGFR, ECAD, c-kit, MGMT, PDGFR, AR, MPR1, SPARC, PTEN, TOP2A, TS,PR, ER, PGP, HER2 and TLE3. Markers analyzed for ovarian cancers includeFISH for HER2, and IHC for TOP2A, TS, PR, ER, PGP, HER2, TLE3, BRCA1,BRCA2, IGFRBP3, IGFRBP4, IGFRBP5, TOPO1, ERCC1 and RRM1. Markersanalyzed for colorectal cancers include sequencing for BRAF and KRAS,and IHC for TOP2A, TS, PTEN and COX2. Markers analyzed for lung cancersinclude FISH for EGFR, EML4-ALK fusion and MET, sequencing for EGFR,BRAF and KRAS, and IHC for TOP2A, PTEN, COX2, TOPO1, ERCC1, RRM1, MPR1,SPARC, BCRP, β-III tubulin, IGFR1 and cMET. Analysis according to the“complete” (e.g., non-origin based) approach include FISH for EGFR andHER2, sequencing for EGFR, c-kit, BRAF and KRAS, and IHC for TOP2A,PTEN, TS, COX2, TOPO1, ERCC1, RRM1, MPR1, SPARC, BCRP, c-kit, MGMT,PDGFR, AR, PR, ER, PGP, and HER2. Additional markers that can beincorporated into biomarker-centric profiles are presented in Table 26.

TABLE 26 Biomarker-centric Profiles DNA Biomarker Gene IHC FISH MutationMA Profile c-Met MET ✓ Lung EML4- EML4, ALK ✓ ✓ Lung ALK Fusion hENT-1SLC29A1 ✓ Ovarian IGFRBP IGFRBP3, ✓ Ovarian IGFRBP4, IGFRBP5 IGF-1RIGF1R ✓ ✓ ✓ Ovarian, Lung MMR MLH1, ✓ Colorectal MSH2, MSH5 p16 CDKN2A ✓Colorectal p21 CDKN1A ✓ p27 CDKN1B ✓ PARP-1 PARP1 ✓ ✓ Ovarian PI3KPIK3CA ✓ ✓ Breast, Ovarian, Colon TLE3 TLE3 ✓ Breast Ovarian

In the therapeutic-centric approach of FIG. 46B, the “complete” panel isperformed to assess all markers without regard to cancer origin. Thepanel includes all markers listed for the biomarker centric panel.

Example 10 Biomarker—Drug Associations

Table 27 lists exemplary associations between biomarkers and drugsassociated with the biomarkers. When the biomarkers are found to beoverexpressed in a patient sample, the drugs are indicated for use intreating the patient as described herein. For each drug, an indicationis given of exemplary techniques that can be used to assess thecorresponding biomarker. One of skill will appreciate that any techniquecan be used as described herein or known in the art, including withoutlimitation microarray, PCR, IHC, ISH, FISH, and/or sequence analysis.Abbreviations in the table include the following: DMA—DNA microrarray;MA—Mutational analysis; IHC—Immunohistochemistry; FISH—Fluorescent insitu hybridization

TABLE 27 Biomarker-Drug Associations Biomarker Drug Associations ABCC1(MRP1) doxorubicin (IHC and DMA), epirubicin (IHC and DMA), methotrexate(IHC and DMA), vincristine (IHC and DMA), vinorelbine (IHC and DMA),vinblastine (IHC and DMA), etoposide (IHC and DMA) ABCG2 (BCRP)cisplatin (IHC and DMA)), carboplatin (IHC and DMA) ADA pentostatin(DMA), cytarabine (DMA) ALK (“EML4- crizotinib (FISH), pemetrexed (FISH)ALK”) AR bicalutamide (IHC and DMA), flutamide (IHC and DMA), abarelix(DMA), goserelin (DMA), leuprolide (DMA), gonadorelin (DMA) ASNSasparaginase (DMA), pegaspargase (DMA) BRCA1 mitomycin (DMA), cisplatin(DMA), carboplatin (DMA) BRCA2 mitomycin (DMA), cisplatin (DMA),carboplatin (DMA) CD52 alemtuzumab (IHC and DMA) CDA cytarabine (DMA)CES2 irinotecan (DMA) DCK gemcitabine (DMA), cytarabine (DMA) DHFRmethotrexate (DMA), pemetrexed (DMA) DNMT1 azacitidine (DMA), decitabine(DMA) DNMT3A azacitidine (DMA), decitabine (DMA) DNMT3B azacitidine(DMA), decitabine (DMA) EGFR gefitinib (FISH and MA), erlotinib (FISHand MA), cetuximab (FISH and MA), panitumumab (FISH and MA) EPHA2dasatinib (DMA) ERBB2 (HER2) trastuzumab (IHC and FISH), lapatinib (IHCand FISH), doxorubicin (FISH), epirubicin (FISH), liposomal-doxorubicin(FISH) ERCC1 cisplatin (IHC and DMA), carboplatin (IHC and DMA),oxaliplatin (IHC and DMA) ER tamoxifen (IHC and DMA), toremifene (DMA),fulvestrant (DMA), anastrozole (IHC and DMA), letrozole (IHC and DMA),exemestane (DMA), aminoglutethimide (DMA), megestrol (DMA),medroxyprogesterone (DMA) FLT1 (VEGFR1) bevacizumab (DMA), sunitinib(DMA), sorafenib (DMA) GART pemetrexed (DMA) HIF1A sunitinib (DMA),sorafenib (DMA) IGFBP3 letrozole (DMA) IGFBP4 letrozole (DMA) IGFBP5letrozole (DMA) KDR (VEGFR2) sunitinib (DMA), sorafenib (DMA) Ki67“tamoxifen + chemotherapy” (IHC) - breast only KIT (cKIT) sunitinib (MAand DMA), sorafenib (DMA), imatinib (MA and DMA), dasatinib (MA and DMA)KRAS gefitinib (MA), erlotinib (MA), cetuximab (MA), panitumumab (MA),sorafenib (MA), combination therapy (VBMCP) (MA) cMET/MET gefitinib(FISH), erlotinib (FISH) MGMT temozolomide (IHC and DMA) PDGFRAsunitinib (DMA), sorafenib (DMA) PDGFRB sunitinib (DMA), sorafenib (DMA)PGP (ABCB1) doxorubicin (IHC and DMA), liposomal doxorubicin (IHC andDMA), epirubicin (IHC and DMA), etoposide (IHC and DMA), teniposide(DMA), docetaxel (IHC and DMA), paclitaxel (IHC and DMA), vincristine(IHC and DMA), vinorelbine (IHC and DMA), vinblastine (IHC and DMA)PIK3CA/PI3K cetuximab (MA), panitumumab (MA), trastuzumab (MA) PRtamoxifen (IHC and DMA), toremifene (DMA), fulvestrant (DMA),anastrozole (IHC and DMA), letrozole (IHC and DMA), exemestane (DMA),aminoglutethimide (DMA), goserelin (DMA), leuprolide (DMA), gonadorelin(DMA), megestrol (DMA), medroxyprogesterone (DMA) PTEN erlotinib (IHC),gefitinib (IHC), cetuximab (IHC), panitumumab (IHC), trastuzumab (IHC)PTGS2 (COX2) celecoxib (IHC and DMA), aspirin (IHC) BRAF1 (BRAF)cetuximab (MA), panitumumab (MA) RARA ATRA (DMA) RRM1 gemcitabine (IHCand DMA), hydroxyurea (DMA) RRM2 gemcitabine (DMA), hydroxyurea (DMA)RRM2B gemcitabine (DMA), hydroxyurea (DMA) RXRB bexarotene (DMA) SPARCnab-paclitaxel (IHC and DMA) (mono/poly) SRC dasatinib (DMA) SSTR2octreotide (DMA) SSTR5 octreotide (DMA) TLE3 paclitaxel (IHC), docetaxel(IHC) TOPO1/TOP1 irinotecan (IHC and DMA), topotecan (IHC and DMA)TOPO2A/TOP2A doxorubicin (IHC, FISH and DMA), liposomal doxorubicin(IHC, FISH and DMA), epirubicin (IHC, FISH and DMA) TOP2B doxorubicin(DMA), liposomal doxorubicin (DMA), epirubicin (DMA) TUBB3 paclitaxel(IHC), docetaxel (IHC), vinorelbine (IHC) TS/TYMS pemetrexed (IHC andDMA), capecitabine (DMA), fluorouracil (IHC and DMA) VDR choleciferol(DMA), calcitriol (DMA) VHL sunitinib (DMA), sorafenib (DMA)

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150024952A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A method of identifying one or more candidate treatment for anovarian cancer in a subject in need thereof, comprising: (a) determininga molecular profile for one or more sample from the subject on a panelof gene or gene products, wherein the molecular profile comprises theresults of assessing the panel of gene or gene products by: performingimmunohistochemistry (IHC) analysis on the one or more sample from thesubject on one or more of: AR, BCRP, CAV-1, CD20, CD52, CK 5/6, CK14,CK17, c-kit, CMET, COX-2, Cyclin D1, E-Cad, EGFR, ER, ERCC1, HER-2,IGF1R, Ki67, MGMT, MRP1, P53, p95, PDGFR, PGP, PR, PTEN, RRM1, SPARC,TLE3, TOPO1, TOPO2A, TS and TUBB3; performing gene expression analysison the one or more sample on one or more of: ABCC1, ABCG2, ADA, AR,ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, cKit, c-MYC,DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERCC1, ERCC3,ESR1-, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HER2/ERBB2,HIF1A, HSP90, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, LCK, LYN, MET, MGMT,MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRa,PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2,RRM2B, RXRB, RXRG, SIK2, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, SPARC,TK1, TNF, TOP2B, TOP2A, TOPO1, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, andZAP70; performing fluorescent in-situ hybridization (FISH) analysis onthe one or more sample on at least one of: ALK, cMET, c-MYC, EGFR,HER-2, PIK3CA, and TOPO2A; and performing DNA sequence analysis or PCRon the one or more sample on at least one of: BRAF, c-kit, EGFR, KRAS,NRAS, and PIK3CA; (b) comparing the molecular profile of the subject toa molecular profile of a reference to identify which of the members ofthe panel are differentially expressed, amplified or mutated as comparedto the reference; (c) accessing a computer database to identify one ormore treatment that is associated with one or more members of the panelthat are differentially expressed, amplified or mutated as compared tothe reference; and (d) providing a computer generated report thatidentifies the at least one drug therapy identified in step c), therebyidentifying the one or more candidate treatment.
 2. (canceled)
 3. Themethod of claim 1, wherein identifying a treatment that is associatedwith one or more members of the panel that are differentially expressed,amplified or mutated as compared to the reference comprises: (i)correlating the one or more members of the panel that are differentiallyexpressed, amplified or mutated with a set of rules, wherein the set ofrules comprises a mapping of treatments whose biological activity isdetermined against cancer cells that have different level of,overexpress, underexpress, and/or have mutations in one or more membersof the panel of gene or gene products; and (ii) identifying the one ormore treatment based on the correlating in (i).
 4. The method of claim3, wherein the set of rules comprises one or more of the rules listed inTable
 5. 5. The method of claim 3, wherein the mapping of treatmentscontained within the set of rules are based on the efficacy of varioustreatments particular for a target gene or gene product.
 6. (canceled)7. The method of claim 1, wherein the one or more sample comprisesformalin-fixed paraffin-embedded (FFPE) tissue, fresh frozen (FF)tissue, a tissue comprised in a solution that preserves nucleic acid orprotein molecules, a core needle biopsy, a bodily fluid, a malignantfluid, a fine needle aspirate (FNA), or a combination of any thereof. 8.(canceled)
 9. (canceled)
 10. The method of claim 1, wherein thereference is from one or more non-cancerous sample.
 11. (canceled) 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. The methodof claim 1, wherein the gene expression analysis comprises using a lowdensity microarray, an expression microarray, a comparative genomichybridization (CGH) microarray, a single nucleotide polymorphism (SNP)microarray, a proteomic array or an antibody array.
 17. (canceled) 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled) 27.(canceled)
 28. The method of claim 1, wherein a prioritized list of theone or more candidate treatments is identified.
 29. (canceled) 30.(canceled)
 31. (canceled)
 32. (canceled)
 33. The method of claim 1,wherein the subject has been previously treated with the candidatetreatment.
 34. The method of claim 1, wherein the subject has notpreviously been treated with the candidate treatment.
 35. The method ofclaim 1, wherein the subject has previously been treated for the cancer.36. The method of claim 1, wherein the cancer comprises a metastaticcancer.
 37. The method of claim 1, wherein the cancer comprises arecurrent cancer.
 38. The method of claim 1, wherein the cancer isrefractory to a prior treatment.
 39. The method of claim 38, wherein theprior treatment comprises the standard of care for the cancer. 40.(canceled)
 41. The method of claim 1, wherein the ovarian cancercomprises an ovarian surface epithelium carcinoma (EOC).
 42. The methodof claim 41, wherein the EOC comprises a surface epithelial tumor,serous cancer, mucinous cancer, endometriod cancer, clear cell cancer,carcinosarcoma, Brenner tumor, cancer of the fallopian tubes, or afemale peritoneal cancer.
 43. The method of claim 1, wherein the ovariancancer comprises a non-epithelium ovarian carcinoma (non-EOC).
 44. Themethod of claim 43, wherein the non-EOC comprises a sarcoma of theovary, malignant germ cell tumor, sex cord-stromal tumor,gonadoblastoma, lymphoma, or other rare tumor of the ovary. 45.(canceled)
 46. (canceled)
 47. (canceled)
 48. The method of claim 1,further comprising determining a prognosis for the ovarian cancer basedon the molecular profile.
 49. The method of claim 48, whereindetermining the prognosis comprises analysis of one or more of cMet,IGF1R, Class III beta tubulin (TUBB3), PIK3CA, Caveolin 1, CK5/6, CK14,CK17, C-kit, c-myc, Cyclin D1, E-cadherin, EGFR, P53, and PDGFR. 50.(canceled)
 51. (canceled)
 52. (canceled)
 53. (canceled)
 54. The methodof claim 1, wherein the subject's progression free survival (PFS),disease free survival (DFS), or lifespan is extended by selection andadministration to the subject of one or more of the one or morecandidate treatment.
 55. (canceled)
 56. A system for carrying out themethod of any previous claim, comprising: a host server; a userinterface for accessing the host server to access and input data; aprocessor for processing the inputted data; a memory coupled to theprocessor for storing the processed data and instructions for: i)accessing the molecular profile generated for the one or more sample;ii) determining which of the members of the panel are differentiallyexpressed, amplified or mutated as compared to the reference; and iii)accessing a rules database to identify one or more agent that interactswith the members of the panel that were determined to be differentiallyexpressed, amplified or mutated as compared to the reference; and adisplay means for displaying the members of the panel that weredetermined to be differentially expressed, amplified or mutated ascompared to the reference and the agents that are associated with them.57. The system of claim 56, wherein the rules database comprises one ormore of the rules in Table
 5. 58. The system of claim 56, wherein therules database comprises the rules in Table
 5. 59. (canceled) 60.(canceled)
 61. (canceled)
 62. (canceled)
 63. (canceled)
 64. (canceled)65. (canceled)
 66. (canceled)
 67. (canceled)
 68. (canceled) 69.(canceled)
 70. (canceled)
 71. (canceled)
 72. (canceled)
 73. (canceled)74. (canceled)